| Objective:Cancer is a common disease threatening human health.According to the incidence and mortality of 36 cancers in 186 countries released by International Agency for Research on Cancer of the World Health Organization in 2020,the number of new cancer patients and cancer deaths in China in 2020 occupy the first position in the world respectively.Due to the aging of the population globally,it is estimated that the burden of cancer will increase by 50% by 2040,and that cancer patients will reach 30 million worldwide.Colorectal cancer(CRC)is one of the most common malignant tumors in the digestive tract.Because of its insidious onset,the cause of CRC is not fully understood.The early treatment of CRC is mainly surgical resection,but because the primary cancer is located in the innermost pelviccavity,the anatomical relationship is more complicated.In the middle and late stages of diagnosis,due to the metastasis of the cancer,surgical resection is no longer possible,and chemotherapy can only be used for treatment.However,most of the chemotherapy drugs currently used in clinical are non-targeted,they kill tumor cells while also affecting normal cell functions.Therefore,it is of great significance to find a specific chemotherapeutic drug that can be used for a long time for the treatment of colorectal cancer.Nuclear factor erythroid-derived 2-related factor 1(NFE2L1)belongs to the CNCb ZIP transcription factor subfamily,which is widely expressed in various tissues and organs including the intestine.Under physiological conditions,NFE2L1 protein is transcribed and translated by mRNA,and it is anchored on the endoplasmic reticulum membrane through its N-terminal transmembrane domain.When no transcription activation signaling,it is deglycosylated and modified by N-glycanase.After turning over into the cytoplasm,it undergoes complete degradation in the endoplasmic reticulum by Ubiquitin proteasome system(UPS),so that maintain a low level in the cytoplasm.When stimulated by oxidative stress,the transcriptional activity of NFE2L1 is activated.At this time,the UPS no longer completely degrades it,but is guided by the DDI2 shuttle factor to cut and modify the N-terminal endoplasmic reticulum transmembrane domain to form the long isoform fragment with transcriptional activity enters the nucleus,forms a heterodimer with the small Maf protein,and finally binds to the antioxidant response element(ARE)in the promoter or enhancer region of the target gene to initiate downstream gene expression,mainly involves a series of genes such as antioxidant response,proteasome homeostasis,oxidative stress,mitochondrial respiration,inflammatory response,and cell proliferation and differentiation.Compared with normal cells,colorectal cancer cells have the characteristics of infinite proliferation,they need to synthesize more biological macromolecules,so the biological operation pressure inside the cells is greater.Protein is an indispensable biological macromolecule to maintain the functioning of the body.UPS is an important mechanism for the degradation of unfolded proteins and the most important protein quality control system in eukaryotic cells.It is currently considered as a new target for the treatment of malignant tumors.The principle is to take advantage of the relatively sensitive characteristics of cancer cells,and by giving cancer patients proteasome inhibitors(PIs)to inhibit the rate of protein degradation,aggravates the biological operation pressure in cancer cells,and ultimately lead to the occurrence of intracellular protein toxicity,which in turn cell death,and then achieving the effect of chemotherapy.Among them,Bortezomib(BTZ)was the first PIs approved by the FDA in 2003,and it is also the current first-line drug for the treatment of hematological tumors.Therefore,this study used lentiviral transfection to obtain different isoforms of NFE2L1 stable knockdown and blank control cell lines.Through in vitro experiments,different time points and different doses of BTZ exposure were performed to explore how it affects the function of the proteasome system and thus affects the colorectal cell lines damage and the role of NFE2L1.Methods:1.Construct control(Scramble)and NFE2L1 all isoform(NFE2L1(A))and long isoform(NFE2L1(L))stable and knockdown colorectal cancer cell lines: 293 T cells are used as viral vectors,and commercialized human NFE2L1 shRNA plasmids are used for packaging.And then the human colorectal adenocarcinoma cell line(Caco-2)is transfected with a lentivirus with a certain viral titer.After 48 hours of infection in the incubator,the replacement contains 1.6 μg/ml puromycin culture medium is screened until there is no large amount of floating cells after passage,and the protein level and mRNA level are verified.2.To determine its inhibitory effect on the proteasome system by acute BTZ exposure:Give different cell lines acute BTZ exposure for 0-24 hr,and quantify NFE2L1 by extracting protein and mRNA.When the UPS function is inhibited,as a substrate to be degraded,the protein degradation rate of NFE2L1 will decrease,so the protein level in the cell will accumulate;at the same time,when the UPS function is inhibited,NFE2L1 will be induced to activate the proteasome subunit expression,so its mRNA level will be activated within a certain range.The relative level of NFE2L1 can reflect the inhibitory effect of BTZ on the proteasome of the Caco-2 cell line.3.To determine whether there are differences in the sensitivity of different cell lines to this cytotoxicity by acute BTZ exposure and by what mechanism: Give different concentrations of BTZ acute exposure 0～24 hours,through CCK8 and trypan blue staining experiments to determine whether there is a difference in cell survival rate between Scramble and NFE2L1(A)-KD and NFE2L1(L)-KD cell lines.Through above experiment,the difference in cell survival rate between Scramble and NFE2L1(A)-KD cell lines was screened,and then some protein of apoptosis and necrosis are tested by Western Blot to consolidate the results.In the part of mechanism analysis,after acute BTZ exposure,the mRNA levels of some proteasome subunits and the related molecules of endoplasmic reticulum stress were detected to preliminarily determine the mechanism through which BTZ induces cytotoxicity and the role of NFE2L1 in it.Result:1.Scramble and stable knockdown cell lines have been obtained through lentiviral transfection,which have been verified by molecular biology.2.Treatment with low concentration of BTZ can cause the accumulation of NFE2L1 in Caco-2 cells,indicating that BTZ has a significant inhibitory effect on the cells.3.After BTZ treatment,NFE2L1(A)cell lines have higher sensitivity to it,but there is no significant difference between NFE2L1(L)and Scramble.4.BTZ treatment can induce cell death mainly due to apoptosis,and NFE2L1(A)deficiency leads to more serious apoptosis.5.BTZ treatment can cause proteasome rebound and stress response in cells.NRF2L1(A)deficiency will inhibit the proteasome rebound mechanism and aggravate the stress response inside the cell.Conclusion: NFE2L1 does not affect the survival and growth of Caco-2 cells.However,NFE2L1(A)cell lines are more sensitive and cause more serious cell death after BTZ treatment.Through the mRNA levels of some proteasome subunits,it was found that NFE2L1(A)can inhibit the rebound mechanism of the proteasome and induce more severe intracellular stress,which is the reason behind the severe apoptosis outcome. |
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