The Role Of RNA Binding Protein ZCCHC4 In Chemotherapeutic Resistance Of Hepatocellular Carcinoma And The Underlying Epigenetic Mechanisms

Hepatocellular carcinoma(HCC)is one of the serious diseases that threaten human health.HCC is characterized of high incidence,poor prognosis and short five-year survival rate.The conventional treatments for HCC include surgical resection,radiotherapy and chemotherapy.Many patients fail to receive surgery resection because of the huge size and poor location of tumor,as well as tumor recurrence and metastasis.For these HCC patients,chemotherapy is the most commonly used therapy.Nowadays,the popular chemotherapeutic drugs contain strogen receptor antagonists,vascular growth factor receptors antagonists,growth factor receptor antagonists,and DNA damage chemical reagents.Disappointedly,these chemotherapeutic drugs are not effective as expected in HCC even using combo treatment.It is still of urgency to explore and reveal the key mechanisms underlying chemotherapeutic resistance in HCC,providing the potential targets for the design of new powerful drugs for the treatment of HCC patients.RNA-binding protein(RBP)is an important player in post-transcriptional modification.It has been widely acknowledged that RBP-RNA interaction networks play an important role in tumorigenesis and tumor progression.Deregulated expression and function of RBPs will influence a variety of cancer hallmarks such as sustained proliferation,metastasis,death resistance,metabolic disorder,immune evasion,genomic instability and so on.Owing to the complexed and important function of RBPs,even small changes of RBP expression and function will result in critical consequences in physiological and pathophylogical processes.There are still many enigmas of RBP-RNA interaction networks in tumors remaining to be explored.ZCCHC4 is a member of the zinc finger protein family which belongs to RBP.Up to now,the function of ZCCHC4 has rarely been reported.Previous study in our lab found that ZCCHC4 was involved in regulating the type I interferon-mediated immune response against hepatitis B virus.So,we hypothesized that ZCCHC4 may play a role in HCC progression.This study aims to investigate the role of ZCCHC4 in HCC development and its underlying mechanism,and to explore whether ZCCHC4 can be a candidate target for adjuvant chemotherapeutic drug.Part ?: The Functional of ZCCHC4 in chemotherapeutic resistance of HCCTo study the role of ZCCHC4 in HCC progression,we tested the expression of ZCCHC4 protein in HCC tissues using human HCC tissue microarrays.We found that the expression of ZCCHC4 in HCC tissues was significantly higher than that in the corresponding adjacent nontumor tissues,and the patients with high ZCCHC4 expression had the significantly shorter overall survival time.Then we constructed three HepG2 cells with ZCCHC4 knock-out(ZCCHC4_KO cell lines)using the CRISPR/Cas9 gene editing technology.We subcutaneously injected HepG2 cells and ZCCHC4_KO cells into nude mice,and found that the tumors of mice bearing HepG2 cells grew faster than that of mice bearing ZCCHC4_KO cells,and the survival time of mice bearing HepG2 cells was much shorter.Thus we came to a conclusion that ZCCHC4 is oncogenic in HCC.Next,we compared the transcriptome difference of HepG2 cells and ZCCHC4_KO cells via RNA sequencing analysis.368 genes were upregulated and 851 genes were downregulated in the ZCCHC4_KO cells compared with that in HepG2 cells.Gene ontology(GO)analysis indicated that the upregulated genes were mainly involved in cell differentiation and cell death,and the downregulated genes were in cell differentiation,cell migration and cell proliferation.KEGG signaling pathway enrichment analysis showed that downregulated genes were mainly involved in PI3K-AKT signaling pathway,p53 signaling pathway,and tumor-associated signaling.The above results implied that ZCCHC4 may be critical in HCC cell survival and viability.Then,we transfected HepG2 cells and SMMC-7721 cells with siZCCHC4,and found that ZCCHC4-silenced HepG2 cells possessed significantly higher apoptosis rate,higher cleaved PARP and cleaved caspase3 signals than those with siNC under oxaliplatin stimulation.And the HepG2 cells with overexpressed ZCCHC4 exhibited the opposite.In addition,the cell killing and growth inhibitory effect of oxaliplatin was more pronounced in ZCCHC4-silenced HCC cells.Moreover,when treated with doxorubicin,the results were the same as that treated with oxaliplatin.Silencing of ZCCHC4 increased the sensitivity of HCC cells to doxorubicin,and overexpression of ZCCHC4 decreased the sensitivity of HCC cells to doxorubicin.Then we constructed HepG2 cell lines that stably overexpressed ZCCHC4 and subcutaneously injected HepG2 cells and ZCCHC4_overexpressed HepG2 cells into nude mice to test the efficiency of oxaliplatin in treatment of HCC in vivo.We found that oxaliplatin significantly inhibited tumor growth in mice bearing HepG2 cells,while had no significant influence on tumor growth in mice bearing HepG2 cells with ZCCHC4 overexpression.Given the above results,we demonstrated that ZCCHC4 contributes to apoptosis resistance in HCC.In order to explore whether the apoptosis-resistance effect of ZCCHC4 was of universal significance,we examined the expression of ZCCHC4 in lung cancer,pancreatic cancer,and colon cancer by tissue microarrays.We found that the expression of ZCCHC4 in tumor tissues of lung cancer,pancreatic cancer and colon cancer was significantly higher than that of corresponding adjacent nontumor tissues.And lung cancer patients and pancreatic cancer patients with higher expression of ZCCHC4 had shorter overall survival time.When lung cancer cell line A549,pancreatic cancer cell line BXPC-3,colon cancer cell line HCT116 were transfected with siZCCHC4,the apoptosis rate induced by oxaliplatin was significantly higher than that with siNC.Taken together,we can conclude that the apoptosis-resistance effect of ZCCHC4 is universal in different human cancer types.Through this part of our study,we confirmed that ZCCHC4 was oncogenic in HCC,and participated in the apoptosis resistance of HCC to oxaliplatin and doxorubicin.The apoptosisresistance effect of ZCCHC4 was of universal significance,since it was also true in lung cancer,pancreatic cancer,and colon cancer.We suggested that ZCCHC4 may serve as a potential target for adjuvant chemotherapy.In-depth mechanism for ZCCHC4 apoptosis-resistance effect needs to be further investigated.Part ?: The mechanism of ZCCHC4 in chemotherapeutic resistance of HCCTo understand the mechanisms for ZCCHC4-mediated apoptosis-resistance effect,we examined the subcellular localization of ZCCHC4 in HCC cells.Western blot and immunofluorescence analysis suggested that ZCCHC4 was mainly localized in the nuclei of HCC cells.Next,we examined the expression of DNA damage-related signals induced by oxaliplatin in HCC cells.We found that the DNA damage-related signals,such as pATM,pATR,pCHK1,pCHK2 and gH2AX,were upregulated in HepG2 cells and SMMC-7721 cells transfected with siZCCHC4 than that with siNC,and the overexpression of ZCCHC4 showed the opposite effect.These results indicated that ZCCHC4 hampers DNA damage process in HCC cells induced by oxaliplatin.Then,we performed co-immunoprecipitation(Co-IP)experiments and found that ZCCHC4 bound directly to gH2AX and the binding intensity increased in HCC cells along with the time of oxaliplatin treatment.Protein mass spectrometry analysis of IP samples further confirmed the interaction of ZCCHC4 and H2 AX in HCC cells once treated by chemical drug.Moreover,the colocalization of ZCCHC4 and gH2AX in HepG2 cells after oxaliplatin treatment for 24 h was detected by immunofluorescence.The above results indicated that ZCCHC4 interacts with gH2AX in HCC under the condition of DNA-damage-induced cell apoptosis.ZCCHC4 contained zinc finger domains and belonged to the RNA-binding protein family,thus we wondered whether there were RNAs pariticating in modulating apoptosis-resistant effect of ZCCHC4.To find the intergenic long non-coding RNAs(lincRNAs)bound by ZCCHC4,we performed PAR-CLIP-seq.After screening of the 107 lincRNAs from PARCLIP-seq analysis,lincRNA AL133467.2 was found to antagonize the apoptosis-resistance function of ZCCHC4.AL133467.2 localized in both cytoplasm and nuclei.So far,there was no report about AL133467.2.Interfering AL133467.2 on the basis of ZCCHC4 silence reduced the sensitivity of HCC cells to oxaliplatin and inhibited the upregulation of gH2AX.Silencing of AL133467.2 alone had no obvious effect on oxaliplatin sensitivity and gH2AX expression.In our study,we utilized RIP assay,RNA pull-down assay,and FISH assay to confirm the presence of the ZCCHC4/AL133467.2/gH2AX complex in the nuclei of HCC cells under the condition of DNA-damage-induced cell apoptosis.Through this part of our study,we found a previously unknown nuclear complex ZCCHC4/AL133467.2/gH2AX complex in HCC cells under the condition of DNA-damageinduced cell apoptosis,which could feedback regulate the DNA damage process induced by chemotherapeutic drugs,and thus affect the chemotherapeutic efficiency.More specific molecular mechanism needs to be further studied.In summary,we found that RNA binding protein ZCCHC4 is oncogenic in HCC,and contributes to the apoptosis resistance of HCC cells to DNA damage reagents.Moreover,the apoptosis resistance function of ZCCHC4 is of universal significance,for it is also true in human lung cancer,pancreatic cancer,and colon cancer.The anti-apoptotic effect of ZCCHC4 is achieved by inhibiting DNA damage process which may be inseparable from the function of the ZCCHC4/AL133467.2/gH2AX nuclei complex.Our findings not only reveal a new nuclear complex associated with DNA damage,which may deepen our understanding of the nuclear complex network in biological process,but also reveal a previously unreported RNA-binding protein which plays an important role in chemotherapeutic resistance,thus providing new mechanistic insight into chemotherapeutic resistance and outlining a new potential target for the combined treatment of HCC in the future.