| Colorectal cancer(CRC)is one of the most common malignant tumors of digestive system,which has a high morbidity and mortality.The treatment of CRC is mainly based on surgery,supplemented by radiotherapy and chemotherapy,but the number of operable cases is usually less than 25%and the tumor recurrence rate is as high as 70%.CRC patients with recurrent or metastatic who cannot be operated on are usually treated with palliative chemotherapy,but it may eventually cause cytotoxic side effects and corrective occurrence,and causing great damage to the patient's immune system,thus causing great pain to the patient.Thus,developing new targets,effective and novel alternative agents and adjuvants with reduced side effects is important to reduce the lethality of the disease and improve the quality of life of patients.As the disease only becomes symptomatic at an advanced stage,worldwide organised screening programmes are being implemented,which aim to increase early detection and reduce morbidity and mortality from colorectal cancer.Numerous studies have confirmed that there is a small group of cells in tumors with strong self-renewal ability and multidirectional differentiation ability.This group of cells is called cancer stem cells(CSCs).Colorectal cancer stem cells(CCSCs)have been associated with the initiation of CRC for a long time.Due to genetic mutations and environmental pressures,the expansion of CCSCs increases.Multiple organs such as liver and lung metastasis may appear at the advanced stage.Increasing evidence has highlighted the role of CCSCs presenting in CRC in aggressiveness,metastasis,chemoresistance and subsequent tumor recurrence.Therefore,understanding the self-renewal and differentiation mechanisms of CCSCs has important clinical significance.Advances in genomic and epigenomic research has shaped our understanding of cancer over the past two decades.Rather than merely a perpetuating mass of dysregulated cells growing in an uncontrolled manner,cancer is also defined by the dynamic genetic and epigenetic alterations that contribute to cancer initiation and progression.Since epigenetic changes such as DNA methylation and histone modifications are crucial factors in developmental programming of stem cells to specific lineages of cellular and tissue differentiation,aberrant epigenetic alterations may transform normal stem cells to cancer stem cells with the loss of differentiation capacity and the acquisition of stem-like characteristics.Accumulating evidence suggests instead that CSCs are a dynamic population continuously shaped by a convergence of genetic,epigenetic,and microenvironmental factors.As epigenetic mechanisms have important functions in modulating stem cell properties in cancer cells,targeting components of these epigenetic pathways would help in eradicating both CSCs and the bulk tumor population.PCGF1,also named as Nervous System Polycomb 1(NSPc1),belongs to the Polycomb Group(PcG)protein family.PcG protein family includes two complexes,PRC 1 and PRC2.PRC 1 is divided into two types,classic PRC1 and non-classical PRC 1 due to different PCGF proteins.The PcG protein family plays an important role in embryonic development and stemness maintenance,and regulates gene transcription and expression through the epigenetic modification of histones.PCGF1 has been shown to inhibit gene transcription activity through H2AK119ub1 and H3K27me3.PCGF1 functions as a key epigenetic regulator of embryonic stem cell self-renewal and early embryogenesis,and is abundantly expressed in several cancers.GEDS and GEPIA database showed that PCGF 1 is generally highly expressed in different types of tumors,PCGF1 promotes tumor cell proliferation and cell cycle progression in cervical cancer cells,malignant gliomas and other tumor cells.In addition,PCGF1 expression is increased in oral squamous cell carcinoma stem cells,PCGF1 promotes self-renewal of glioma stem cells.However,the role of PCGF1 in CRC and CCSCs remains largely unknown.Under the above research background,we carried out the following experiments to study the regulatory role of PCGF1 in CRC and CCSCs.Part ? PCGF1 promotes CCSCs enrichment and proliferationWe analyzed the expression of PCGF1 in a variety of solid tumors using GEDS and GEPIA database,and the results showed that PCGF1 was significantly upregulated in cancer tissues compared with normal controls.This is consistent with the results that reported in previous studies.However,whether PCGF1 affect the occurrence and development of CRC remains to be determined.The first part we analyzed the role of PCGF1 in CRC and CCSCs,and obtained the following results:(1)GEDS and GEPIA databas were used to analyze the expression of PCGF1 in CRC tumor tissues and that in adjacent tissues,and the results showed that the expression level of PCGF1 was significantly higher in CRC tissues compared with normal controls.(2)We used the GEPIA and Kaplan-Meier Plotter database to analyze the correlation between the expression of PCGF1 and patients' overall survival(OS),and the results showed that the expression level of PCGF1 was negatively correlated with the OS of CRC patients.The OS of patients with high PCGF1 expression is significantly reduced(P<0.05).We further analyzed the correlated between the expression of PCGF1 and the grade of tumor malignancy(?-?),and the results showed that the expression level of PCGF1 was positively correlated with the clinical stage of CRC,that is,the expression level of PCGF 1 increased with the increase of tumor malignancy.We further detected the expression level of PCGF 1 in normal intestinal epithelial cells HCoEpiC and a panel of CRC tumor cell lines using RT-qPCR and Western Blot.The results showed that PCGF1 mRNA and protein was highly expressed in tumor cells than in HCoEpiC.(3)A PCGF1 loss-of-function subclone was generated in HCT116 cells,which have high endogenous PCGF1 expression,using a pLKO-PCGF1 shRNA lentivirus,and a PCGF1 gain-of-function subclone was generated in SW620 cells,which have low endogenous PCGF1 expression,using a pLVX-PCGF1 lentivirus.Then we induced HCT116-CSCs and SW620-CSCs,and detected the expression level of PCGF1 in tumor cells and CCSCs using RT-qPCR and Western Blot.The results showed that PCGF1 mRNA and protein was highly expressed in CCSCs than tumor cells.(4)We tested the effect of PCGF1 on the enrichment of CCSCs,and found that PCGF1 knockdown strongly inhibited tumour sphere formation of HCT116 cells;the tumour spheres decreased in size,and the number of spheres(diameter?50 ?m)notably decreased,Conversely,compared to vector control cells,SW620 cells overexpressing PCGF1 robustly promoted colorectal cancer stem cell sphere formation;the tumor spheres became larger and the number of spheres(diameter?50?m)was increased.(5)We tested the effect of PCGF 1 on CCSCs proliferation using Ki-67 staining and CCK-8 assay,and the results showed that cell proliferation was enhanced by PCGF1,while cell proliferation was reduced after PCGF1 knockdown.Western Blot and AV-PI staining were used to verify the effect of PCGF 1 on CCSCs apoptosis,and the results showed PCGF1 had no significant effect on cell apoptosis.(6)Balb/c nude mice tumorigenesis assays indicated that PCGF1 knockdown significantly inhibits tumour growth in vivo.Part ? PCGF1 regulates signaling pathways related to CCSCs selfrenewalThe first part showed that PCGF1 promotes the enrichment of CCSCs and proliferation.In order to explore the mechanisms,we performed the following exploration and obtained the following results:(1)First,we investigated the relationships between PCGF1 and Wnt signaling pathway key proteins WNT3A,WNT6,LRP6,WNT8A and ?-catenin,and Notch pathway key proteins NOTCH1,NOTCH3.The correlation between PCGF 1 and the above proteins as follows:PCGF 1 between WNT3A(R=0.65,P=0),WNT8A(R=0.78,P=0),WNT6(R=0.31,P=1.5e-10),LRP6(R=0.5,P=0),?-catenin(R=0.59,P=0),NOTCH1(R=0.57,P=0),NOTCH3(R=0.35,P=2.8e-13)in primary CRC tissue samples in the GEPIA database.The correlation between PCGF1 and Wnt signaling pathway is higher according to the correlation coefficient.(2)We further detected the expression of ?-catenin through RT-qPCR and Western Blot experiments,and the results showed that PCGF1 knockdown significantly reduced the expression of ?-catenin.On the other hand,PCGF1 overexpression significantly increased the expression of ?-catenin.Part ? The epigenetic regulation mechnism of PCGF1 on the proliferation of colorectal cancer stem cellsPCGF1 is an important epigenetic regulatory factor,and it has been proveded that PCGF1 could inhibit gene transcription activity by regulating H3K27me3.Whether PCGF1 affects the CCSCs enrichment by regulating the level of histone epigenetic modification?After the second part,we futher tested the influence of PCGF1 on the epigenetic regulation of CCSCs.We performed the following mechanism exploration and obtained the following results:(1)First,we investigated the relationships between PCGF1 and CCSCs stemness markers like CD133,CD44,LGR5,SOX2 and ALDH1A1.The correlation between PCGF1 and the above stemness markers as follows:PCGF1 between CD 133(R=0.17,P=0.0027),CD44(R=0.38,P=1.7e-12),SOX2(R=0.19,P=0.00092),ALDH1A1(R=0.23,P=3e-05),LGR5(R=0.34,P=6e-10)and OCT4(R=0.49,P=0)in primary CRC tissue samples in the GEPIA database.We further examined the regulatory effect of PCGF1 on the expression of stemness markers using RT-qPCR,Western Blot analyses and immunofluorescence staining,and the results were consistent with the prediction of database.(2)We detected the level of epigenetic modification at the whole cell level using Western Blot,and the results showed that the expression levels of H3K4me3 was decreased while the H3K27me3 was increased after PCGF1 knockdown.When PCGF1 overexpression,the expression levels of H3K4me3 was increased while the H3K27me3 was decreased.The ChIP-qPCR assays were used to determine whether PCGF1 regulates CCSCs markers expression through histone modifications,and the results showed that the level of H3K4me3 was significantly decreased while the level of H3K27me3 increased at the promoter of CD133,CD44 and ALDH1A1 after PCGF1 knockdown.(3)Next,we examined histone modification machinery involving in H3K4me3 and H3K27me3 modifications using RT-qPCR and immunofluorescence staining,and the results showed that both H3K27me3 demethylase KDM6A and H3K4me3 methyltransferase KMT2A decreased significantly,but H3K4me3 demethylase KDM5C increased after PCGF1 knockdown;Both the expression levels of KDM6A and KMT2A were increased,KDM5C decreased after PCGF1 overexpression.ConclusionPCGF1 was significantly upregulated in colorectal cancer tissues compared with normal controls.In addition,PCGF1 expression was higher in advanced malignant tumors and significantly associated with the clinical stage(Stage I to IV).PCGF1 overexpression promoted CCSCs enrichment and proliferation,and the in vivo results further confirmed the results of in vitro.In terms of mechanism,on the one hand,PCGF1 is positively correlated with key proteins of stemness-related signaling pathways and regulated the expression of ?-catenin in Wnt signaling pathway;On the other hand,PCGF1 regulated the expression of CCSCs' markers through histone modifications,and PCGF1 regulated histone methylation modification by regulating the expression of histone methylase and demethylase.Our findings suggested PCGF1 as a potential therapeutic target for CRC treatment.Innovation:1.PCGF1 is upregulated in CRC;The expression of PCGF1 was higher in advanced malignant tumors and significantly associated with the clinical stage;PCGF1 mRNA levels greater than the median which were associated with decreased overall survival(OS)rates.2.PCGF1 promotes CCSCs enrichment and proliferation in vivo and in vitro.3.PCGF1 promotes the enrichment of CCSCs by regulating signaling pathways which related to CCSCs self-renewal and the histone methylation level at the promoter region of stemness markers.Our research will help to provide a promising therapeutic target of CRC. |
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