| Background:Colorectal cancer(CRC)is a malignant tumor that seriously endangers human health and is the most common digestive system tumor.At present,the treatment for CRC is mainly comprehensive surgical treatment.Although the treatment strategy for CRC has made significant improvement and progress in recent years,the prognosis and five-year survival rate of advanced patients have not reached satisfactory results due to the high metastasis rate and high recurrence rate of CRC.Therefore,identifying and screening biomarkers for the occurrence and development of CRC,finding new intervention targets,studying its biological behavior,and clarifying its mechanism of action have always been the critical directions of CRC research and carrying out basic and clinical research related to CRC has essential clinical application and transformation value.Recent studies have shown that the occurrence and development of tumors are closely related to mitochondria.Mitochondria produce the energy needed by the body through oxidative phosphorylation.Changes in this process or mitochondrial dysfunction in biosynthesis,cell differentiation,cell apoptosis,signal transduction,and so on that mitochondria participate in can lead to cancer.The inner membrane mitochondrial protein(IMMT),also known as MIC60/Mitofilin,is the core component of the assembly and maintenance of the mitochondrial contact site and cristae organizing system(MICOS)and participates in the control of biological processes such as the morphology of mitochondrial cristae,protein transport,mitochondrial DNA transcription,and the inner and outer membranes connecting mitochondria.Recent studies have shown that the role of IMMT in lung cancer,gastric cancer,liver cancer,pancreatic cancer,prostate cancer,ovarian cancer,and other tumors has attracted attention.Relevant studies mainly involve the biological characteristics of tumor cell lines or the pathological parameters of tumor patients.There is a lack of systematic research on the role and mechanism of IMMT in tumors,and research in CRC has yet to be reported.Based on the above background,this study proposed the scientific hypothesis that IMMT may participate in the occurrence and development of CRC by regulating mitochondrial homeostasis and carried out the following research with IMMT as the starting point.Objective:To clarify the differential expression and biological role of IMMT in CRC tissue,observe the biological behavior of IMMT on the proliferation,migration,invasion,and other biological behaviors of colorectal cancer cells and the changes of mitochondrial homeostasis of cells,explore the relevant mechanism of IMMT in the occurrence and development of colorectal cancer and provide new ideas and targets for clinical diagnosis,treatment and intervention of colorectal cancer.Methods:1.Using liquid chromatography-mass spectrometry to detect and analyze the mitochondrial protein of colorectal cancer tissue and colorectal cancer/adjacent tissues by proteomics and establish the differential protein expression profile;2.Bioinformatics technology was used to analyze the relationship between the biological function,pathway,and expression of IMMT in CRC and the prognosis of patients;3.Western blot,RT-q PCR,and immunohistochemistry were used to verify the expression of IMMT in CRC cancer/adjacent tissues and observe the relationship between the expression of IMMT and the pathological parameters of patients;4.Western blot and RT-q PCR were used to detect the protein and m RNA expression of IMMT in common colorectal cancer cell lines;The silent IMMT(sh IMMT)HT29 cell line and the overexpression IMMT(ov IMMT)HCT116 cell line were constructed by liposome transfection technology,and the subcutaneous tumorigenic model of sh/ov IMMT was constructed in nude mice;5.The proliferation of HCT116,HT29 cells,and transplanted tumor after sh/ov IMMT were detected by the CCK8 method,clone formation test,the growth curve of the transplanted tumor,and Ki-67 staining;Scratch test,Transwell test,Western blot,and immunofluorescence staining were used to verify the migration,invasion ability and expression of essential proteins in epithelial-mesenchymal transition(EMT)of sh/ov IMMT colorectal cancer cells and transplanted tumors;The apoptosis and cell cycle of sh/ov IMMT colorectal cancer cells were detected by flow cytometry and Tunel staining;6.The stable transfected sh/ov IMMT cell line and the nude mice subcutaneous tumorigenic model were constructed.The ROS level and mitochondrial membrane potential of sh/ov IMMT colorectal cancer cells were detected by immunofluorescence,and ELISA detected the changes of intracellular ATP content;Western blot was used to detect the expression of mitochondrial mitotic fusion proteins OPA1,MFN1,MFN2 and DRP1 in colorectal cancer cell lines and metastatic tumors;7.RNA sequencing technology was used to screen the differential genes of the HT29 colorectal cancer cell line ov IMMT,and the complement C3 was selected as the research target.The liposome transfection technology was used to construct sh NC,sh IMMT,sh IMMT+ov NC,and sh IMMT+ov C3 colorectal cancer cell lines.Western blot was used to verify the transfection efficiency and construct the subcutaneous tumorigenic model of sh IMMT+ov C3 in nude mice;8.CCK8 method,clone formation test,the growth curve of the transplanted tumor,and Ki-67 staining were used to detect the proliferative ability to interfere with IMMT and complement C3 on colorectal cancer cells and transplanted tumor;the Scratch test,Transwell test,Western blot,and immunofluorescence method were used to observe the migration and invasion ability of IMMT and complement C3 to CRC cells,and the expression of EMT key protein was detected;The effects of interfering IMMT and complement C3 on apoptosis and cell cycle of colorectal cancer cells and transplanted tumor were detected by flow cytometry and Tunel staining;9.The stable transfected sh IMMT+ov C3 cell line and the subcutaneous tumorigenic model of nude mice were constructed.The ROS level and mitochondrial membrane potential of sh IMMT+ov C3/ov NC colorectal cancer cells were detected by immunofluorescence staining,and ELISA detected the changes of intracellular ATP content;Western blot was used to detect the expression of mitochondrial mitotic fusion proteins OPA1,MFN1,MFN2 and DRP1 in colorectal cancer cell lines and metastatic tumors.Results:1.A total of 1549 mitochondrial proteins were identified by mitochondrial proteomics analysis and compared with the differentially expressed protein profile of colorectal cancer/paracancerous tissue.IMMT was differentially expressed in total protein/mitochondrial protein and cancer/paracancerous tissue of colorectal cancer;2.Bioinformatics analysis showed that IMMT was distributed and expressed in14 normal human tissues and 18 joint tumor tissues.The expression level of colorectal-derived cell lines was high(P<0.05),and it was highly expressed in the cytoplasm;IMMT was highly expressed in 12 joint tumor tissues,including colon cancer,and correlated with the poor prognosis of CRC patients(P<0.05);3.Western blot,RT-q PCR,and immunohistochemistry methods verified that the high expression of IMMT in colorectal cancer tissue was positively correlated with clinical staging and tumor size(P<0.05),which played a role in promoting cancer;4.Western blot and RT-q PCR showed that IMMT was expressed in colorectal cancer cell lines,relatively high in HT29 cell lines,and relatively low in HCT116 cell lines(P<0.05);The colorectal cancer cell line sh/ov IMMT and the subcutaneously transplanted tumor in nude mice were successfully constructed;5.CCK-8 and plate cloning showed that the proliferative capacity of colorectal cancer cells in the ov NC group was significantly lower than that in the ov IMMT group(P<0.05);The subcutaneously transplanted tumor in nude mice showed that the volume of transplanted tumor in the ov IMMT group was significantly increased(P<0.05),and the proliferation of colorectal cancer cells was enhanced;Scratch and Transwell test showed that the migration and invasion ability of cells in ov NC group was significantly weaker than that in ov IMMT group(P<0.05),and Western blot and immunofluorescence showed that the expression of crucial EMT proteins N-cadherin,Vimentin,and Snail in ov IMMT group increased(P<0.05);Flow cytometry and Tunel staining showed that apoptosis in ov IMMT group was significantly lower than that in ov NC group(P<0.05),and the number of cells in S phase increased;6.Immunofluorescence showed that the mitochondrial membrane potential and ROS level of the ov IMMT group were increased,and ELISA showed that the intracellular ATP level of the ov IMMT group was increased(P<0.05);Western blot showed that compared with ov NC,the expression of mitochondrion division and fusion proteins OPA1,MFN1,MFN2,and DRP1 in colorectal cancer cells and transplanted tumors in ov IMMT group increased(P<0.05);Compared with sh NC,the mitochondrial membrane potential and ROS level of cells and transplanted tumors in sh IMMT group decreased,ATP production decreased(P<0.05),mitochondrial division and fusion protein expression decreased(P<0.05);7.RNA sequencing showed that the complement C3 gene was differentially expressed in ov IMMT HT29 colorectal cancer cells;Successfully constructed sh NC,sh IMMT,sh IMMT+ov NC,and sh IMMT+ov C3 cell lines and subcutaneously transplanted tumors in nude mice;8.CCK-8 and plate cloning showed that the proliferation of colorectal cancer cells in the sh IMMT+ov NC group was significantly lower than that in the sh IMMT+ov C3 group(P<0.05);The subcutaneously transplanted tumor in nude mice showed that the volume of transplanted tumor in sh IMMT+ov C3 group was significantly increased(P<0.05),and the proliferation of colorectal cancer cells was enhanced;Scratch and Transwell test showed that the migration and invasion ability of cells in sh IMMT+ov NC group was significantly weaker than that in sh IMMT+ov C3 group(P<0.05).Western blot and immunofluorescence showed that the expression of crucial EMT proteins N-cadherin,Vimentin,and Snail in the sh IMMT+ov C3 group increased(P<0.05);Flow cytometry and Tunel staining showed that the apoptosis of sh IMMT+ov C3 group was significantly lower than that of sh IMMT+ov NC group(P<0.05),and the number of S phase cells increased;9.Immunofluorescence showed that compared with the sh IMMT+ov NC group,the mitochondrial membrane potential and ROS level of cells in the sh IMMT+ov C3 group increased,and the level of intracellular ATP in sh IMMT+ov C3 group increased(P<0.05);Western blot showed that compared with sh IMMT+ov NC,the mitosis of colorectal cancer cells and the expression of fusion proteins OPA1,MFN1,MFN2,and DRP1 were increased in sh IMMT+ov C3 group(P<0.05).Conclusions:1.IMMT is highly expressed in the mitochondria of CRC cancer tissues and colorectal cancer cells,related to the occurrence and development of colorectal cancer.The prognosis is poor when the expression level is elevated,suggesting that it has the role of promoting cancer.2.IMMT affects the biological processes of colorectal cancer cells,enhancing their proliferation,migration,invasion,and anti-apoptotic abilities.3.IMMT affects the mitochondrial homeostasis of colorectal cancer cells by regulating energy metabolism,redox process,mitochondrial fission,and fusion of colorectal cancer cells.4.IMMT-complement C3 regulates mitochondrial homeostasis and affects biological behaviors such as proliferation and invasion of colorectal cancer cells,which may be one of the mechanisms by which IMMT promotes the occurrence and development of CRC. |
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