The Role And Mechanism Of MB Regulated By MPC1 On Maintaining "Stemness" Of Non-small-cell Lung Cancer

BACKGROUND ＆ OBJECTIVE: Lung cancer is one of the malignant tumors with the highest morbidity and mortality worldwide,which poses a major threat to population health and life safety.Non-small-cell lung cancer（NSCLC）accounts for about80% of all lung cancers.Cancer stem cells（CSCs）,also known as tumor-initiating cells（TICs）,are cells with stem-like biological characteristics（hereinafter referred to as "stemness"）present in tumor tissues.Cancer stem cells may be involved in malignant biological behaviors such as tumor cell renewal,recurrence and metastasis,tumor nutrient angiogenesis,and increased tolerance to chemoradiotherapy after their occurrence.Therefore,it is important to study the maintenance mechanism of "stemness" of lung cancer stem cells（LCSCs）and target LCSCs for reducing the drug resistance of tumors,inhibiting their metastasis and recurrence,and improving the quality of life of patients.Mitochondrial pyruvate carrier（MPC）is an important protein that plays an important role in the process of pyruvate crossing the mitochondrial membrane and is located on the mitochondrial inner membrane.Human MPC is a complex composed of MPC1 as well as MPC2,and the heterodimer composed of both regulates the flux of pyruvate into the mitochondrial matrix thereby regulating energy metabolism.Numerous studies have shown that MPC1 expression is low in lung cancer,and that the expression level is significantly correlated with lung cancer prognosis.METHODS: In this study,dedifferentiated LCSCs spheres were enriched as LCSCs model to explore the changes in their metabolism,the expression level of MPC1 and the changes in intracellular pyruvate content,and the effects of supplied pyruvate on the expression level of MPC1 and the changes in the "stemness" level of LCSCs were explored,and the expression changes of MPC1 during LCSCs differentiation were explored by serum differentiation.Herein,NSCLC cell lines stably knocked down and overexpressing MPC1 were established by lentiviral transfection to verify the effects of knockdown and overexpression of MPC1 on the "stemness" of NSCLC cells and malignant biological behaviors such as proliferation,invasion,and migration.Furthermore,myoglobin（MB）,a molecule that can interact with MPC1,was found by protein profiling,and the interaction between the two and the subcellular localization of MPC1-MB complex was demonstrated by co-immunoprecipitation.Finally,we focused on the level of cellular oxidative stress,the expression levels of mitochondrial superoxide and DNA damage markers in LCSCs were detected based on the biological function of MB.RESULTS: 1.Low expression of MPC1 in LCSCs:（1）LCSCs are in increased autophagy level;（2）LCSCs are in a chronic hypoxic state,and MPC1 expression is significantly low in LCSCs;（3）With the extension of serum differentiation time,the expression level of MPC1 in LCSCs generally increases,and MPC1 is involved in the regulation of LCSCs differentiation.2.MPC1 affects the invasion,metastasis and proliferation of NSCLC cells:（1）After knockdown of MPC1,the invasion and migration of H1299 and H1975 cells are enhanced,and the results are opposite after overexpression of MPC1;（2）Knockdown of MPC1 enhances the colony formation rate of H1299 and H1975 cells,enhances the cell proliferation ability,and has the opposite results after overexpression of MPC1;（3）After knockdown of MPC1,the expression of N-cadherin and Slug,which are related indicators of epithelial-mesenchymal transition（EMT）,is upregulated,E-cadherin expression is down-regulated,and the cells undergo epithelialmesenchymal transition,and the results are opposite after overexpression of MPC1.3.MPC1 is involved in the maintenance of "stemness" in NSCLC:（1）After knockdown of MPC1,the expression levels of cell "stemness" markers SOX2 and CD133 were significantly up-regulated,and the above markers were significantly down-regulated after overexpression;（2）Sphere formation assay showed that after knockdown of MPC1,the diameter and number of tumor cell spheres were significantly higher than those of the control group,and overexpression of MPC1 inhibited the sphere-forming ability of cells.4.MPC1 regulates MB to maintain NSCLC "stemness" :（1）Co-IP experiments confirmed that MPC1 interacts with MB;（2）After knockdown of MPC1,MB expression is downregulated in mitochondria,nuclear translocation is increased,MB nuclear expression is up-regulated in LCSCs,and MPC1 affects MB intracellular localization;（3）High MB nuclear expression protects against the attack of intracellular superoxide on nuclear genetic material,and MPC1 regulates MB nuclear translocation to affect DNA damage repair in LCSCs.CONCLUSION: In LCSCs,chronic hypoxic environment and high expression of MB inhibit the production of mitochondrial superoxide,and low expression of MPC1 reduces the mitochondrial translocation of MB through the interaction with MB,and simultaneously mediates MB entry into the nucleus,resists the attack of intracellular superoxide on genetic material in the nucleus,promotes the escape of LCSCs,and causes tumor recurrence and metastasis.Targeting MPC1 to reduce CSCs "stemness" and promote CSCs differentiation is expected to be a new target for cancer therapy.