| SKA2(Spindle and Kinetochore Associated Complex Subunit 2),also known as FAM33A(Family with Sequence Similarity 33,Member A),It is a subunit of the spindle and kinetochore associated complex(SKA),which plays an important role in cell cycle regulation and the development of cancer and other diseases.We previously found that SKA2 expression was significantly up-regulated in lung cancer tissues and was involved in regulating the proliferation,migration and invasion of lung cancer cells,but its specific molecular mechanism remains unclear.This study aims to further explore the molecular mechanism of SKA2 promoting the proliferation and migration of lung cancer cells.(1)SKA2 expression was significantly up-regulated in lung cancerWe analyzed the expression of SKA2 in lung cancer tissues from the TCGA data set using the GEPIA database,and the results showed that SKA2 expression was significantly up-regulated in lung cancer tissues compared with the control normal lung tissues,and the expression level of SKA2 was negatively correlated with the survival rate of lung cancer patients.(2)Expression profile of SKA2 gene silencing in lung cancer cell line A549We performed further bioinformatics technical analysis of the microarray data published by Rice et al.that silenced SKA2 gene in lung cancer cell A549.For SKA2 leads to gene silencing the differentially expressed genes of clustering and KEGG gene function analysis,the results showed that the enrichment significantly differentially expressed genes in transport,regulation of transcription and Oxidation-reduction process such as biology,at the same time hints SKA2 could lead to gene silencing PDSS2 key enzyme involved in the biosynthesis of metabolic pathways,such as gene expression.It is also suggested that SKA2 gene silencing may lead to up-regulation of PDSS2 and other key enzymes involved in the biosynthetic metabolic pathway.SKA2 expression in lung cancer A549 cells was inhibited by RNA interference.The RT-PCR results showed that SKA2 gene silencing could lead to the down-regulation of ABCA1 gene and significantly up-regulation of PDSS2 gene expression.PDSS2 is the first enzyme involved in CoQ10 biosynthesis pathway and also the key enzyme,which is worthy of further study.(3)SKA2 inhibited the expression of PDSS2 and decreased the content of CoQ10To further confirm whether SKA2 regulates the expression of PDSS2 and the synthesis of CoQ10,we constructed a SKA2-overexpressing plasmid pcDNA3.0-SKA2 and silenced SKA2 using RNA interference technology.RT-PCR and Western Blot analysis showed that the expression of PDSS2 was significantly decreased and the level of CoQ10 b was decreased after SKA2 overexpression,while the expression of PDSS2 was significantly increased and the level of CoQ10 b was increased after SKA2 interference.These data suggest that SKA2 inhibits the expression of PDSS2 gene at m RNA and protein levels,and subsequently inhibits the synthesis of CoQ10.In order to confirm whether SKA2 can regulate PDSS2 gene transcription,we co-transfected PDSS2 promoter tructs and SKA2-overexpressing plasmid pcDNA3.0-SKA2 into lung cancer cell A549 to detect the activity of PDSS2 gene promoter.The results showed that SKA2 inhibited the activity of several promoter truncates,including the core promoter of PDSS2.These results indicated that SKA2 inhibited the expression of PDSS2 gene at the transcriptional level.(4)Effect of CoQ10 on proliferation and migration of lung cancer cellsWe first analyzed the effects of CoQ10 on the proliferation and migration of lung cancer cell A549.The proliferation of lung cancer cell A549 was observed by adding 50μM and 100μM CoQ10,respectively.The results showed that CoQ10 had no significant effect on the proliferation of A549 cells.At the same time,the scratch healing experiment results showed that CoQ10 had no significant effect on the migration ability of lung cancer cell A549.(5)Effect of CoQ10 on proliferation and migration of colorectal cancer cellsNext,we further analyzed whether CoQ10 regulates the proliferation and migration of other cancer cells,such as colorectal cancer cells SW480 and RKO.The results showed that CoQ10 had no significant effect on the proliferation,cell cycle,apoptosis and cell migration of colorectal cancer SW480 cells,and had no significant effect on the proliferation of RKO,but could inhibit the migration of RKO.(6)Preliminary study on the molecular mechanism of CoQ10 inhibiting RKO migration in colorectal cancer cellsWe used RNA-seq technique to analyze the changes of RKO gene expression profile induced by CoQ10 treatment in the scratch healing experiment.GO and KEGG analysis showed that the differentially expressed genes were significantly enriched in cell adhesion molecules signaling pathways,extracellular matrix receptor signaling pathways and steroid hormone biosynthesis signaling pathways.Quantitative RT-PCR verification results showed that CoQ10 treatment could significantly up-regulate the expression of PCSK9,HMGCR,HMGCS1 and DHCR7 genes in the cholesterol synthesis pathway and MMP10 gene in the extracellular matrix pathway.These results suggest that CoQ10 may inhibit RKO migration in colorectal cancer cells by regulating the expression of these genes.In conclusion,based on previous studies,the molecular mechanism of SKA2 promoting the proliferation and migration of lung cancer cells was initially explored in this study,and it was found that SKA2 could inhibit the expression of PDSS2 and possibly inhibit the synthesis of CoQ10,laying a solid foundation for further exploration of the role of SKA2 in the development and development of lung cancer.At the same time,we also found that CoQ10 may inhibit the migration ability of colorectal cancer cells by affecting cholesterol metabolism,which has important theoretical and practical significance for further exploring the molecular mechanism and therapeutic value of CoQ10 in lung cancer,colorectal cancer and other malignant tumors. |
