Study On The Role And Mechanism Of ZCCHC7 In The Proliferation Of Colorectal Cancer

Background and ObjectiveColorectal cancer(CRC)is characterized by high incidence rate and mortality,which causes the second largest cancer death in the world.In general,effective surgical treatment or adjuvant radiotherapy and chemotherapy is the main treatment strategy for CRC,but the prognosis of combined treatment is generally poor.According to a large number of current reports,bufalin has a wide range of anti-tumor acti vity.Using bufalin as a molecular probe,high-throughput protein microarray screening and analysis indicated that Zinc finger CCHC domain-containing protein 7(ZCCHC7)may be involved in the regulation of malignant progression of CRC.ZCCHC7 is a member of the zinc finger CCHC domain-containing protein family,and it’s an RNA-binding protein.However,the relationship between ZCCHC7 and malignancy progression and its specific regulatory mechanism in malignancy are still poorly understood,especially in CRC.In this study,we intend to investigate the role of ZCCHC7 in CRC proliferation in vitro and in vivo,to investigate the specific mechanisms by which ZCCHC7 affects CRC cell proliferation,and to provide new molecular targets for CRC clinical treatment.Methods1.The potential molecular target ZCCHC7 was screened by co-incubation of labeled-bufalin probes and protein chip.The survival curves of CRC patients with high expression of ZCCHC7 and those with low expression of ZCCHC7 were analyzed by GEO clinical database to explore the relationship between ZCCHC7 expression and survival rate of CRC patients.2.Stable cell lines overexpressing or knockdown ZCCHC7 were constructed based on lentiviral transfection in two CRC cell lines,RKO and HT29.Western blot(WB)was used to verify the effect of overexpression and knockdown of ZCCHC7.Proliferation rates of CRC cells after overexpression and knockdown of ZCCHC7 was detected by CCK-8 and colony formation assay.Apoptosis of CRC cells after inducible knockdown of ZCCHC7 by Tetracycline was detected based on Flow cytometry.To investigate the tumorigenicity of ZCCHC7 in vivo,a subcutaneous xenograft model was constructed in NOD/SCID mice.3.RNA-seq sequencing,RIP-seq sequencing and protein profiling were used to explore the target protein and mRNA downstream of ZCCHC7.Small interfering RNA(siRNA)targeting the downstream target protein was designed and transfected into CRC cells,WB assay was used to detect the knockdown efficiency.Detection of the effect of knockdown target proteins on CRC cell proliferation was employed using CCK-8 and colony formation assay.Coimmunoprecipitation(Co-IP)assay was used to measure the interaction between ZCCHC7 and downstream proteins,and the nuclear localization of downstream proteins after overexpression and knockdown of ZCCHC7 was detected by nucleo-cytoplasmic separation assay.The colocalization of ZCCHC7 with downstream protein and the nuclear localization of downstream protein after overexpressing ZCCHC7 were detected by immunofluorescence assay.4.MTT assay was used to verify the effect of bufalin on the proliferation of CRC cells,and WB assay was used to detect the effect of bufalin on the expression of ZCCHC7 protein.The effect of bufalin on apoptosis of CRC cells was verified by apoptosis experiment.Results1.The protein chip demonstrated the binding between labeled bufalin and ZCCHC7.Analysis of clinical CRC-related databases revealed that ZCCHC7 was highly expressed in CRC patients,which was closely associated with poor prognosis of CRC patients.2.The WB and CCK-8 experiments showed that overexpression of ZCCHC7 promoted the proliferation of CRC cells,while knockdown of ZCCHC7 inhibited the proliferation of CRC cells.The results of apoptosis experiments showed that knockdown of ZCCHC7 promoted CRC cell apoptosis.The data of xenograft tumor model showed that overexpression of ZCCHC7 significantly promoted the growth of CRC tumors.3.The results of sequencing analysis suggested that SRSF1 may be involved in the process of ZCCHC7 promoting CRC cell proliferation and was associated with alternative splicing events.Further analysis of RIP-seq sequencing suggested that SRSF1 might be involved in the regulation of ATG16L1 alternative splicing.The effect of siRNA on inhibiting SRSF1 was significant and the results of CCK-8 experiment and colony formation assay showed that knockdown of SRSF1 inhibited CRC cell proliferation.The results of Co-IP experiment showed that ZCCHC7 interacted with SRSF1,which can recruit SRSF1 into nucleus.The nuclear-cytoplasmic separation experiments showed that overexpression of ZCCHC7 increased the nuclear localization of SRSF1,while knockdown of ZCCHC7 decreased the nuclear localization of SRSF1.Moreover,the nuclear localization of SRSF1 was significantly inhibited after treatment with nuclear import inhibitor.The results of immunofluorescence assay showed that ZCCHC7 colocalized with SRSF1,and overexpression of ZCCHC7 increased the nuclear localization of SRSF1.4.MTT assay showed that bufalin could effectively inhibit the proliferation of CRC cells.WB assay found that bufalin could repress the expression of ZCCHC7 protein in CRC cells.Furthermore,the results of apoptosis experiment showed that bufalin could promote the apoptosis of CRC cells.ConclusionThis study demonstrates the role of ZCCHC7 in promoting CRC proliferation.The related mechanism of ZCCHC7 in regulating CRC progression is that it increases the nuclear localization of SRSF1 by recruiting it into the nucleus,which causes the alternative splicing of ATG16L1 and ultimately promotes the proliferation of CRC cells.In addition,bufalin,known as the monomer of traditional Chinese medicine decreases CRC proliferation by inhibiting the expression of ZCCHC7.This study aims to provide a new molecular target for the clinical diagnosis and treatment of CRC.