NCAPD2 Promotes The Growth Of Colorectal Cancer Cells And Inhibits The Molecular Mechanism Of Autophagy

In this study,we employed colorectal cancer(CRC)tissues and adjacent normal tissues,CRC cells and normal colonic mucosal epithelial cell as experimental materias.By using experimental methods and techniques of molecular biology,cell biology and biochemistry,we explored the important role of NCAPD2(subunit of chromosome condensed protein complex I)in colorectal cancer cells and its molecular mechanism of promoting colorectal cancer cell growth and inhibiting autophagy.The results are shown as follows: 1.NCAPD2 expression was upregulated in the CRC tissues and CRC cellsThe expression of NCAPD2 in CRC tissues and adjacent normal tissues was detected by transcriptome sequencing,IHC staining,qRT-PCR and western blotting.The results showed that expression of NCAPD2 was higher in CRC tissues and cells than that in adjacent normal tissues and normal cells.These results suggested that NCAPD2 might play an important role in the development of CRC.2.NCAPD2 facilitated the transition of cell cycle and resulted in promoting the growth of CRC cellsNCAPD2 expression plasmid and siRNA were transfected into HCT116 and SW480 cells respectively.The effects of NCAPD2 on cell growth and proliferation were evaluated by EdU labeling assay,colony formation,cell cycle and apoptosis assays;and western blot assay was employed to check the expression of related genes.The data showed that the proportion of EdU-positive cells and the number or size of cell clones were markedly increased in NCAPD2-overexpression group compared to the control group.Further,NCAPD2 overexpression upregulated the expression of CCND1 and CCNE1 and downregulated the expression of P27,whereas NCAPD2 knockdown had the reverse results and partially induced cell apoptosis.These results suggested that NCAPD2 promoted the growth of CRC cells by promoting the transition of cell cycle.3.NCAPD2 promoted cell growth by activating mTORC1-S6 K pathway in CRC cellsNCAPD2 expression plasmid and siRNA were transfected into HCT116 and SW480 cells respectively,and then the activation of mTORC1 pathway was detected by western blot assay.The data showed that NCAPD2 overexpression increased the levels of p-mTOR(S2448),p-p70S6K(T389/412)and p-4E-BP1(T70).whereas NCAPD2 knockdown had the reverse effects.When cells treated with 3nM rapamycin for another 24 hr after 24 hr of transfection,we found that NCAPD2 knockdown partially increased the rapamycin-induced decrease of p-p70S6K(T389/412).These results suggested that NCAPD2 promoted the growth of CRC cells by activating mTORC1 pathway.4.NCAPD2 inhibited autophagy and blocked autophagic flux by inhibiting mTORC1-ATG pathwayNCAPD2 expression plasmid and siRNA were transfected into HCT116 and SW480 cells respectively.The mTOR and autophagy-related genes were detected by western blot.Immunofluorescent staining of LC3 and P62 were also used to evaluate the effect on autophagy and autophagic flux.The results showed that NCAPD2 overexpression upregulated the level of p-mTOR(S2448),decreased the expression of Beclin1,ATG5,LC3 II and increased the expression of P62;whereas NCAPD2 knockdown had the reverse effects.It is suggested that NCAPD2 inhibited cell autophagy and blocked autophagic flux in CRC cells.Summary: Our studies demonstrated that NCAPD2 was highly expressed in CRC tissues and cell lines.NCAPD2 activated mTORC1 and then facilitated the transition of cell cycle,inhibited cell autophagy and blocked autophagic flux,and finally resulted in promoting the growth and proliferation of CRC cells.