SKA3 Promotes The Stemness Of Hepatocellular Carcinoma Cells

Purpose: Hepatocellular carcinoma(HCC)has high incidence and mortality.There are many reasons leading to its poor prognosis,such as loss of surgical treatment opportunity due to invasion and metastasis,chemotherapy resistance,and postoperative recurrence and metastasis.There are some tumor cells having stem cell characteristics such as self-renewal,differentiation and permanent proliferation,which significantly increase the risk of invasion,metastasis,drug resistance and recurrence of HCC.Therefore,clarifying the molecular mechanism of liver cancer stem cells involved in the recurrence and metastasis of HCC,and looking for new intervention targets,may shed new light to the HCC targeted treatment.Spindle-mitochondrial-associated protein complex subunit 3(SKA3)is subunit of the spindle-mitochondrial-associated protein complex and is essential for cell mitosis.Recently,one study showed that SKA3 can promote the proliferation of HCC cells,but its effect on the stemness characteristics of HCC and the mechanism have not been reported yet.Therefore,we want to observe whether SKA3 can affect the stemness of HCC cells and explore its molecular mechanism by this study.Methods: Firstly,we analyzed the expression difference of SKA3 in HCC tissues and adjacent tissues by analyzing the TCGA database and Western blot of 32 pairs tissue proteins,and performed clinical prognosis correlation analysis through bioinformatics;(2)We used si RNA to down-regulate SKA3,and used SKA3 plasmid to up-regulate SKA3 in HCC cells.Then we observed the stemness biological behavior changes of HCC cells by sphere formation assay,Sorafenib resistance experiment,plate cloning experiment,CCK8 experiment and Transwell migration invasion assay;(3)We used viruses to knockdown or overexpress SKA3 in MHCC-97 h.Then we performed nude mouse subcutaneous xenograft experiment using limited dilution method to further verify the effect of SKA3 on the HCC cells stemness biological behavior in vivo;(4)Finally,to explore the molecular mechanism,first we used Western blot to find the changes of the key molecules of Notch signaling pathway such as Notch1,NICD in HCC cells after down-regulation or up-regulation of SKA3;then we detected the effect of SKA3 on the proliferation and migration abilities of HCC cells after using lentivirus to knock out Notch1 in MHCC-97 h.Results:(1)We found that SKA3 was highly expressed in HCC tissues compared with adjacent tissues.And it was closely related to the survival time and clinical pathological stage of HCC patients;(2)After down-regulating SKA3 expression in HCC cells MHCC-97 h and SNU-398,the results showed that compared with the NC group,the self-renewal ability(SNU-398: P <0.01),Sorafenib resistance ability(MHCC-97h: P <0.0001;SNU-398: P <0.001),colony-forming ability(MHCC-97h: P <0.001;SNU-398: P <0.01),proliferation ability(MHCC-97h: P <0.0001;SNU-398: P <0.0001),migration ability(MHCC-97h: P <0.01;SNU-398: P <0.001),and invasion ability(MHCC-97h: P <0.001;SNU-398: P <0.01)of the si SKA3 group were all significantly weakened.While up-regulating SKA3 in HCC cells,these abilities were enhanced and the results were statistically significant;(3)The subcutaneous xenograft model nude mice showed that in MHCC-97 h,the volume of subcutaneous tumors of the SKA3 depletion group is smaller than the control group(P<0.05),while the SKA3 overexpression group had larger volume of subcutaneous tumors than the control group(P<0.05);(4)Western blot showed that in HCC,the expression of key molecules of the Notch signaling pathway such as Notch1,NICD and Hes1 decreased obviously when SKA3 was down-regulated,and increased obviously when SKA3 was up-regulated.After inhibiting Notch signaling pathway,the promotion of SKA3 on the proliferation ability(P <0.01)and migration ability(P <0.01)of MHCC-97 h was inhibited.Conclusion: Our study showed that SKA3 was highly expressed in HCC and was associated with poor prognosis.In vivo and in vitro experiments had confirmed that SKA3 could promote the stemness of HCC obviously.And preliminary mechanism studies suggested that the promotion effect might be achieved through the Notch signaling pathway.It provided a potential molecular target for supressing HCC recurrence,metastasis and chemotherapy resistance from the perspective of blocking the stemness of HCC cells.