Sp2 Promotes Invasion And Metastasis Of Hepatocellular Carcinoma By Targeting TRIB3 Protein

Background Hepatocellular Carcinoma(HCC)is one of the most common malignant tumors in clinical practice,with morbidity and mortality ranked high among all malignant tumors.Due to the insidious onset,high invasiveness and easy metastasis,most of the patients were already in the advanced stage at the time of diagnosis,thus losing the opportunity of radical resection.Although there has been some progress in the treatment of primary liver cancer in recent years,its long-term survival is still unsatisfactory.Therefore,it is very important to explore the molecular basis behind the invasion and metastasis of primary liver cancer.A large number of studies have shown that continuously activated endoplasmic reticulum stress(ERS)is closely related to the occurrence and development of tumors.Under endoplasmic reticulum stress,three pathways of unfolded protein response(UPR)were activated by three transmembrane proteins that include IRE1,PERK and ATF6.It can not only protect tumor cells from body killing by repairing damaged proteins or degrading misfolded proteins,but also regulate the growth of tumor cells,promote tumor invasion and metastasis through various ways,and mediate chemotherapy resistance.Under the action of endoplasmic reticulum stress,tumor cell apoptosis can be induced.Under endoplasmic reticulum stress,TRIB3 was up-regulated as a sensor of cell stress.TRIB3,a member of the mammalian tribbles family of pseudokinases,plays a key role in the unfolded protein response,is involved in tumor progression,and is associated with poor prognosis.Sp2 is one of the transcription factors of the SP family.Previous results show that SP may play a key role in the emergence,evolution and metastasis of tumors.Most research mainly explores the effects of Sp1 in human cancer.Sp1 is a crucial transcription factor that regulates the growth of cancer cells.Its abnormal expression regulates the proliferation and metastasis of tumors through complex mechanisms.However,there are few reports on the relationship between Sp2 and cancer.Recent studies have shown that Sp2 may be overexpressed in gastric cancer and prostate cancer and play an important role in promoting cancer,while the role of Sp2 in liver cancer has not been studied.Therefore,the purpose of this study was to investigate the relationship between the expression of Sp2 in liver cancer and its clinicopathological characteristics,to further clarify its influence on the proliferation,apoptosis and invasion of liver cancer cells,and to explore its possible molecular mechanism.Objective 1.To explore the expression of Sp2 in HCC and its relationship with clinicopathological features.2.To elucidate the role of Sp2 in proliferation,invasion,migration and apoptosis of HCC cell lines.3.To further explore the possible molecular mechanism of the pro-cancer effect of Sp2.Methods 1.Tissue microarray was prepared from 95 HCC patients with histopathologically confirmed.The expression of Sp2 in HCC tissues and paracancer tissues was detected by immunohistochemistry(IHC),and the correlation between Sp2 and the clinicopathological characteristics was further analyzed.Meanwhile,Sp2 Protein levels in 4 pairs of fresh liver cancer tissues and adjacent tissues were detected by Western Blot method.2.The expression of Sp2 in Hep G2,Huh7 and Hep3 B cell lines was detected by Western Blot method.Hep G2 and Huh7 cell lines with relatively high Sp2 expression were selected for follow-up experiments.Cell proliferation,migration,invasion and apoptosis were detected by CCK8,clone formation test,wound-healing assay,transwell and flow cytometry.3.The expression of GRP78 in the 95 primary liver cancer tissues and adjacent nontumor tissues was detected by immunohistochemistry,and detect the GRP78 protein express in 4 pairs of fresh liver cancer/adjacent liver tissues by Western Blot.Differences in Sp2 expression between high ERS group(high GRP78 expression)and low-ERS group(low GRP78 expression)were analyzed.Sp2 was knocked out in HCC cells by si RNA transfection,and the expression changes of GRP78,ATF6,PERK and IRE1 were detected by Western Blot.4.Chromatin Immunoprecipitation(Chip-seq)was analyzed to predict the possible target genes.Immunohistochemical method was used to detect the expression of possible target gene TRIB3 in the above 95 cases of primary liver cancer tissues and adjacent tissues,and the expression of TRIB3 in Sp2 high / low expression groups was further analyzed.Sp2 was knocked out with small interfering RNA,and Western Blot method was used to detect the expression of TRIB3 protein after Sp2 interference.At the same time,small hairpin RNA was used to interfere with the TRIB3 protein,and the transwell method was used to detect the migration and invasion of liver cancer cells.Results 1.Immunohistochemical results showed that Sp2 was highly expressed in liver cancer samples(52/95)but significantly lower in paracancer tissues.The clinicopathological analysis showed that Sp2 expression was independent of age,gender and histological grade of patients,and was positively correlated with tumor size(P=0.041),lymph node metastasis(P=0.032)and clinical stage(P=0.011).Survival analysis suggested that HCC patients with high SP2 expression had a poor prognosis.Western Blot indicated that Sp2 was significantly overexpressed in liver cancer tissues compared with the corresponding paracellular tissues.The TCGA liver cancer genome database supports this conclusion.Kaplan-meier survival analysis of TCGA data also showed a positive correlation between high Sp2 expression and shorter total survival time.2.CCK8 and clone formation test showed that the deletion of Sp2 in liver cancer cells inhibited cell proliferation and colony formation.wound-healing assay,transwell test showed that knockdown of Sp2 could significantly inhibit the migration and invasion ability of HCC cells.In addition,flow cytometry results also showed that Sp2 knockout in HCC cell lines could significantly increase apoptosis.3.Immunohistochemical and Western Blot results showed that GRP78 was highly expressed in liver cancer tissues.And in 95 liver cancer tissue samples,we found that Sp2 has stronger expression in the group of high ERS.In vitro experiments showed that Sp2 knockdown significantly down-regulated the expression of GRP78.In addition,Knockout of SP2 significantly reduced the protein level of ATF6 but not PERK and IRE1.4.The immunohistochemical results proved that the expression of TRIB3 in HCC cancer tissues was higher than that in adjacent tissues.TRIB3 showed a higher expression trend in high GRP78 and Sp2 groups,respectively.The results of Western Blot showed that Sp2 silencing could down-regulate the expression of TRIB3 in HCC cells.The migration and invasion ability of Hep G2 and huh7 cell lines were both inhibited after TRIB3 knockout.Conclusion 1.SP2 is overexpressed in liver cancer and its expression is associated with HCC progression and poor prognosis.2.Sp2 knockdown inhibit the proliferation,migration and invasion of HCC cells,and promote cell apoptosis.3.Sp2 knockout down-regulates endoplasmic reticulum stress,especially the inhibition of ATF6 signaling pathway.But PERK and IRE1 weren’t lowered.4.Transcription factor SP2 may act on TRIB3 protein and enhance its expression,further regulating endoplasmic reticulum stress biological process through ATF6 signaling pathway.