Effects And Mechanisms Of Endoplasmic Reticulum Stressrelated Transcription Factor ZNF263 On Apoptosis Resistance Of Hepatocellular Carcinoma Cells

Background Hepatocellular carcinoma(HCC)is one of the leading causes of cancer death worldwide.The 5-year survival rate of liver cancer,including HCC,is just about 18%.The main reason for the poor clinical treatment results of patients with advanced HCC is still the apoptosis resistance of tumor cells to chemotherapy drugs.Therefore,more research is urgently needed to explore effective treatment strategies for HCC.Endoplasmic reticulum stress(ERS)is an important factor affecting the sensitivity of liver cancer drugs.In addition to changing the biological characteristics of tumor cells,endoplasmic reticulum stress can significantly reduce the sensitivity of liver cancer cells to chemotherapy drugs.However,the mechanism by which endoplasmic reticulum stress induces tumor cell resistance is not completely clear,involving multiple genes,multiple proteins,and multiple signaling pathways,which makes it very difficult to formulate related treatments.Endoplasmic reticulum stress-mediated autophagy is also an important factor affecting the sensitivity of liver cancer drugs.Inhibition of autophagy can increase the sensitivity of liver cancer drugs.However,the mechanism by which endoplasmic reticulum stress regulates autophagy is not fully understood.Super enhancers(SEs)are a hot spot in tumor research at present.Carcinogenic super enhancers exist in many cancer subtypes and are essential for maintaining the characteristics of cancer cells.Compared with ordinary enhancers,super enhancers span a larger genomic region and are enriched with more histone modifications related to transcription factors,cofactors and transcriptional activity.Super enhancers drive the expression of key oncogenes and confer tumor dependence,which ultimately provides a therapeutic idea for cancers that lack a well-known genetic driver and primary secondary resistance to tumors.However,the relationship between super enhancers and their target genes,transcription factors,ERS and autophagy,and the sensitivity to cancer drugs have rarely been reported.Since super-enhancers are more dependent on the transcription factors they bind to than ordinary enhancers,this enlightens us that at the transcription level,we can use the relevant transcription factors that interfere with super-enhancers as a starting point to suppress oncogenes are activated in the process of apoptosis resistance.It has been reported that zinc finger protein 263(ZNF263)is upregulated in HCC.However,its functional role in liver cancer remains to be studied.In the current study,we found that ZNF263 is activated in endoplasmic reticulum-stressed cells,upregulated in HCC patients and cell lines,and is related to drug sensitivity of HCC cells,but its relationship with autophagy has not been reported.It may be a potentially target for HCC treatment.Objective To explore transcription factors that might bind super-enhancers that were potentially associated with ERS were found in HCC cells.To explore the effects of this transcription factor on phenotypic changes such as proliferation and apoptosis resistance of HCC cells and changes in drug sensitivity and its underlying mechanisms.Methods(1)Transcriptome sequencing was used to identify ER stress related m RNAs.(2)Histone-modified H3K27 ac Ch IP-seq combined with transcriptome sequencing was used to identify the potentially related super-enhancers of HCC cells after ERS,superenhancers of HCC cells after ERS induction,and the transcription factors of ERS potentially related SEs were further analyzed by MEME software.(3)Tissue chip technology was used to explore the expression of ZNF263 in cancer and adjacent tissues of patients with hepatocellular carcinoma.Survival curve analysis the relationship between ZNF263 and the overall survival of patients with hepatocellular carcinoma,the relationship between ZNF263 and intracellular stress marker protein GRP78,and the autophagy marker protein Beclin1,LC3.(4)RNAi knockdown of ZNF263,CCK8,and flow cytometry was used to observe the changes of HCC cell proliferation,anti-apoptotic ability and drug sensitivity.(5)Immunofluorescence,Western blotting,electron microscopy,and immunohistochemistry were used to observe the changes in the autophagy level of HCC cells in which RNAi interfered with ZNF263.(6)Western blotting,flow cytometry and immunofluorescence techniques was used to investigate the expression of ZNF263,autophagy levels,and sensitivity of HCC cells to sorafenib in HCC cells under endoplasmic reticulum stress induced by tunicamycin(TM)or inhibited 4-phenylbutyric acid(4-PBA).(7)RNAi knocked down ZNF263,the autophagy level of HCC and the sensitivity of HCC cells to sorafenib under ERS were observed by Western blotting,electron microscopy and flow cytometry.Results(1)Transcriptome sequencing identified ER stress-related m RNAs.302 genes were significantly up-regulated,and 183 genes were significantly down-regulated(?Log2FC??1.5,p <0.05).(2)H3K27ac Ch IP-seq found 197 new super-enhancers in HCC cells after ERS,and intersected with transcriptome-upregulated genes to obtain 17 ERS potentially related super-enhancers and their regulated target genes.The MEME software was used to predict SEs-related transcription factors and found that four transcription factors such as ZNF263 may bind to ERS related SEs.(3)Tissue microarray results showed that ZNF263 expression was up-regulated in liver cancer tissues,which was related to the high expression of ERS marker protein GRP78 and shortened overall survival of HCC patients.(4)RNAi knocked down ZNF263,which increased the proliferation and anti-apoptotic capacity of HCC cells,reduced the sensitivity of HCC to anticancer drugs.(5)ZNF263 was knocked down by RNAi,and Western blotting and electron microscope data showed that autophagy levels of hepatocellular carcinoma cells were reduced.At the same time,immunohistochemical data showed that the high expression of ZNF263 was related to the high expression of autophagy marker proteins Beclin1 and LC3.(6)Western blotting,immunofluorescence and flow cytometry data showed that in tunicamycin-induced HCC cells,endoplasmic reticulum stress activated ZNF263 and reduced the drug sensitivity of HCC cells.A specific inhibitor 4-PBA that used to inhibit endoplasmic reticulum stress inhibited ZNF263 and autophagy,and increased the drug sensitivity of HCC cells.(7)RNAi knockdown of ZNF263,Western blotting and electron microscopy data showed that autophagy level of HCC cells under ERS was down-regulated,and drug sensitivity of HCC cells was showed increased by flow cytometry.This suggested that ZNF263 might increase the apoptosis resistance of HCC cells by activating autophagy associated with ER stress in HCC cells.Conclusions(1)ZNF263 may be one of the transcription factors bound by ERS potentially related super-enhancers.It is up-regulated in patients with hepatocellular carcinoma and is associated with poor prognosis in patients with hepatocellular carcinoma.(2)ZNF263 promotes the proliferation and apoptosis resistance of HCC cells and reduces the sensitivity of HCC cells to sorafenib.(3)ERS is an upstream event of autophagy.ERS activates the expression of ZNF263,which in turn up-regulated the level of autophagy in HCC cells,and participates in the process of decreasing the sensitivity of ERS HCC cells to sorafenib treatment.Therefore,ZNF263 may be one of the key mechanisms of ERS-mediated autophagy to cause hepatocellular carcinoma cells apoptosis resistance.