CHD1L Promotes The Autophagy And Migration Of Hepatocellular Carcinoma Cells Via Targeted Inhibition Of ZKSCAN3 Transcription

Background and purposeThe amplification and overexpression of CHD1 L gene exist in many malignant tumors.It plays an important role in the invasion and metastasis of tumor cells and is closely associated with the malignant progression,staging and poor prognosis of tumors.Identification of CHD1 L as a target to block the progression of malignant tumors has potential clinical applications.The premise is to fully interpret the role of CHD1 L and its molecular regulation mechanism.By analyzing our previous CHD1 L ChIP-sequencing results,a zinc finger family DNA-binding protein,key suppressor gene of autophagy,ZKSCAN3,emerged as a CHD1L-interacitve molecule.Potential binding between CHD1 L and ZKSCAN3 suggests that CHD1 L may be involved in the regulation of autophagy.Autophagy(macroautophagy)is a catabolic process that removes aggregated proteins and damaged organelles in cells.Two opposite functions of autophagy are implicated in maintaining cellular homeostasis and development of cancers.On one hand,autophagy may function as a tumor suppressor by preventing the accumulation of dysfunctional mitochondria to decrease oxidative stress,DNA damage whereas it may also function as a tumor promoter to alleviate metabolic stress during tumorigenesis and facilitate cell spreading,migration,and invasion in highly metastatic tumor cells.Overexpressed ZKSCAN3 is reported to be associated with the proliferation,migration and invasion in colorectal cancer,multiple myeloma and prostate cancer,however,some other publications also proved a significant decrease in the expression levels of ZKSCAN3 in upper urinary tract urothelial carcinoma(UUTUC).All of those studies indicate a scenario-dependent role of ZKSCAN3 in tumor cells,leaving a possibility for that there may be an unknown upstream molecule or mechanism that affects the function of ZKSCAN3.Analysis of CHD1L-binding DNA sequences by ChIP-Sequencing suggested that the upstream pathway of ZKSCAN3 was still worth investigating.Explaining the roles and mechanisms between CHD1 L and ZKSCAN3 is critical for understand the role of CHD1 L in promoting tumor progression and its relationship with autophagy regulation.MethodsChIP-PCR and ChIP-qPCR assay were applied to verify the binding region of CHD1 L to indicated DNA;Transfection of liposomes establishes transient overexpression of CHD1 L or downregulation of ZKSCCAN3,Paxillin,and ATG5 cell lines;Co-immunoprecipitation(Co-IP)method for detection of protein-protein binding;immunoblotting detection of related protein expression;real-time fluorescence quantitative PCR for detection of related gene transcriptional levels;transmission electron microscopy for intracellular autophagy or autophagy lysosomes;immunofluorescence assays were applied to detect the formation of autophagosomes and subcellular co-localization of two protein;transwell and wound-healing assays to detect cell migration.Results1、CHD1L activates autophagy via inhibiting ZKSCAN3 transcription.By analyzing our previous ChIP-sequencing results,a key suppressor gene of autophagy,ZKSCAN3,emerged as a CHD1L-interacitve molecule.ChIP-PCR was performed to validate the potential binding of CHD1 L to the indicated region at 4741 to4341 bp upstream to the TSS of ZKSCAN3.Results suggested in line with the ChIP sequencing result,CHD1 L could bind to the region at 4692 to 4341 bp upstream to the TSS of ZKSCAN3.Furthermore,forced expression of CHD1 L caused a significant decrease in ZKSCAN3 m RNA and protein expression when compared with its vector control.On the contrary,downregulation or depletion of CHD1 L could reversely increase the expression of ZKSCAN3.Immunofluorescence(IF)staining showed a reduced fluorescence intensity of nuclear localized ZKSCAN3 in CHD1 L positive cells that transfected with CHD1L-GFP constructs,relative to the cells with weak expression of CHD1 L.Western blot analysis for a cohort of 10 HCC tissue specimens and corresponding para-tumor tissues showed that ZKSCAN3 expressed in both the tumor and para-tumor tissue,whereas,a lower tendency was found in tumor tissues compared to its para-tumor tissues,which was oppositely related to that of CHD1 L.Recent study has identified ZKSCAN3 as a pivotal suppressor of autophagy and lysosome biogenesis transcriptional.We presumed that autophagy and lysosome biogenesis should be regulated by CHD1 L.The effect of upregulation or downregulation of CHD1 L on both the autophagosome and autophagy flux markers LC3-II and P62 basic levels were firstly detected in HCC cells.LC3-II was elevated in 7402-CHD1 L cells when compared with its vector control,whereas downregulation or deletion of CHD1 L by si RNA or CRISPR/Cas9 approach significantly decreased LC3-II expression in Huh7 and QGY-7703 cells,respectively,and the P62 levels were oppositely with that of LC3-II.To determine whether these alteration of LC3-II levels resulted from autophagosome biogenesis or autophagy flux,we inhibited autophagosome-lysosome fusion using bafilomycin A1(labeled as Baf A1),Results showed that the Baf A1 increased the expression level of LC3-II in HCC cells,especially in CHD1 L overexpressed cells,however,only a slight elevation could be found in CHD1 L downregulation or knocking out cells,Baf A1 could not increase the LC3-II levels in Huh7 or 7703 cells after abolishing CHD1 L expression.Accordantly,the P62 levels were oppositely with that of LC3-II,no significant changes were found in the quantity of P62 induced by Baf A1 treatment after silencing or knocking out of CHD1 L.We assessed whether CHD1 L was important for the starvation-induced autophagy response.Similar with other studies,serum starvation induced a directed increase in LC3-II level,but was significantly blocked by CHD1 L ablation.Moreover,autophagy-lysosome inhibitors(Baf A1 and CQ)has no additive effects with the decrease of LC3-II caused by CHD1 L ablation under starvation.Immunofluorescence analyses for LC3 B showed that LC3 B puncta formation could not be found in 7703-CHD1L-i KO cells after Dox induction,and Baf A1 treatment had no significant effects on puncta reversion under starvation(8 h)or normal circumstances.Transmission electron microscopy was performed to identify autophagic vacuoles,result showed that the number of autophagic vacuoles per cell decreased significantly in Dox induced CHD1L-knocking out cells when compared with that of control.To test further whether CHD1 L acts through ZKSCAN3 to promote autophagy,we first detected the downstream target genes of ZKSCAN3 in 7703-CHD1L-i KO cells.Consistent with ZKSCAN3,the expression levels of these downstream genes were significantly changed with deleting CHD1 L.To investigate whether ZKSCAN3 downregulation could rescue CHD1L-dependent autophagy regulation,we then transfected 7703-CHD1L-i KO cells with ZKSCAN3-1 si RNA.In line with our imagination,knocking down of ZKSCAN3 completely rescued LC3-II enrichment with or without Baf A1 treatment in the given conditions to control levels,as monitored by LC3-II.As shown in immunofluorescence analyses,silencing of ZKSCAN3 increased LC3-B-positive autophagic puncta number remarkably in CHD1L-deletion cells under normal or starved conditions,which was even more significant in the presence of Baf A1.2、CHD1L enhances cell migration by mediating Paxillin autophagic degradationDifferent autophagy inhibitors were used to verify the effect of autophagy on the migration of QGY-7703 and Huh7 cells by in vitro transwell-based migration assay and wound-healing assay.As it has been reported,no matter what kind of autophagy inhibitors were used,the ability of cell migration was significantly inhibited in both QGY-7703 and Huh7 cells when compared with the solvent control.Given that autophagy exerts a similar impact on HCC migration as CHD1 L which has been well-addressed in the progression of HCC,and that,we have just mentioned above,CHD1 L is critical for autophagosome formation,we speculated that migration of tumor cells modulated by CHD1 L may depend on autophagy.To this end,two different autophagy inhibitors,Baf A1 and CQ,were used to observe the effects of CHD1 L on cell migration after blocking the autophagy.Both transwell-based migration assay and wound-healing assay showed that,CHD1 L had no significant effects on migratory capacity of the cells when autophagy was blocked by either Baf A1 or CQ.These results indicated that autophagy was required for CHD1 L mediated migration.It is proved that the key FA protein Paxillin is degraded by autophagy through directly interacting with LC3 at its conserved LC3 binding region(LIR).Co-immunoprecipitation of LC3-B and Paxillin analysis confirmed the binding of endogenous Paxillin with endogenous LC3-B in QGY-7703 cells.Knocking out CHD1 L increased Paxillin accumulation,whereas there was no significant changes on Paxillin m RNA.However,both CQ and Baf A1 had no significant effect on Paxillin accumulation when with or without Dox induction for deleting CHD1 L.Similarly,Paxillin was not controlled by CHD1 L in cells lacking of Atg5,the classical autophagy associated protein.As a result from CHD1 L associated autophagic degradation of Paxillin,it was necessary to determine whether this degradation was related to CHD1 L regulated ZKSCAN3 expression.Knocking down ZKSCAN3 in 7703-CHD1L-i KO cells decreased Paxillin accumulation when compared with control group in the given conditions,implicated that CHD1 Lpromoted autophagic degradation of Paxillin required ZKSCAN3.Previous studies have demonstrated that inhibition of autophagy increases FA size and number due to impaired FA disassembly instead of FA assembly.To identify whether CHD1 L has a role in FA dynamics,immunofluorescence staining for Paxillin and Zyxin was applied,and the results indicated an increased FA size after CHD1 L deletion,meaning for that CHD1 L affected the dynamics of FA.In order to gain further insight into whether Paxillin accumulation will affect the CHD1 L promoted migration,both the transwell-based migration and the wound-healing assay indicated that cell migration ability was reversed by Paxillin-si RNA after knocking out of CHD1 L,compared with that of Paxillin-si RNA control.Furthermore,we performed immunofluorescense at the same conditions to clarify whether FA turnover was promoted by Paxillin-si RNA.Shown as results,the size of FA was decreased when knocked down Paxillin.Altogether,these results indicated that dowregulation of Paxillin could facilitate FA disassembly and subsequently reverse the impairment of tumor cells migration following with CHD1 L deletion.Conclusion:1.CHD1 L binds to the region within at-4692 bp to-4341 bp upstream to the TSS of ZKSCAN3 and represses the transcription of ZKSCAN3.2.Reduction of ZKSCAN3 will relieve its suppression to the transcription of downstream autophagy-related genes,and therefore enhances autophagy.3.Enhanced autophagy accelerates the degradation of Paxillin which interacted with processed LC3 and the focal adhesion will be disassembled to expedite the tumor cell migration.