The Mechanism Of ZFAS1 Promoting RRNA 2'-O-methylation Mediated CRCs Proliferation By Targeting NOP58

Objective:Colorectal cancer?CRC?is the third most malignant tumor and the first digestive tumor in the world,and the incidence and mortality of CRC remain high.According to data from the National Cancer Center of China,the mortality rate of CRC in China is increasing year by year.More importantly,it is rising among young adults.The etiology of CRC is complicated in human and multifactor involved in carcinogenesis including environmental exposures and lifestyle factors,multiple inherited genetic variations and epigenetic modification,etc.In recent years,long non-coding RNA?lncRNA?is not the"dark matter"of the human genome by virtue of its powerful ability to regulate genetic material and plays an important role as oncogene or tumor suppressor in transcription,post-transcriptional,epigenetic modification and translation in a variety of cancers.ZFAS1 is a novel lncRNA,which is involved in the occurrence and development of various cancers as an oncogene,and as well as a diagnostic and prognostic biomarker.So ZFAS1 plays a key role in regulating the development of tumors.It's interesting that ZFAS1 hosts three small nucleolar RNAs?snoRNA?,including SNORD12,SNORD12B and SNORD12C,which belong to C/D box snoRNA.They function by 2'-O-methylation to rRNA-specific sites.Most importantly,C/D box snoRNAs must interact with four proteins,NOP58,NOP56,Fibrillarin,and SNU13 which are evolutionarily stable,highly conserved,to form a functional small nucleolar ribonucleoprotein complexes?snoRNP?to achieve the 2'-O-methylation.rRNA 2'-O-methylation can regulate translational activity and translation ferdility.Thus,it is very important to study the mechanism of snoRNP key proteins.NOP58,one of the key proteins of C/D box snoRNP,has been reported to play an important role in the regulation of acute myeloid leukemia.Whether ZFAS1 can mediate the molecular mechanism of rRNA 2'-O-methylation by targeting NOP58 to promote the malignant biological behavior of colorectal cancer cells requires further experimental verification.Methods:1.Cell model construction HCT116 and SW620 were transfected with pcDH-ZFAS1 and pcDH-NOP58 overexpression plasmid or shZFAS1#1,shZFAS1#2,shNOP58#1 and shNOP58#2 silencing plasmid;antisense oligonucleotide ASO-SNORD12C/78 to conduct the differential expression model of colorectal cancer cell.2.qRT-PCR and Western Blot qRT-PCR and Western Blot were used to detect RNA and protein expression in the cell model and CRC tissues.3.Cell phenotype assay Cell proliferation,migration,and apoptosis were detected by CCK-8,transwell and flow cytometry,respectively.4.mRNA stability analysis SW620 cells were transfected with shZFAS1#1 to knockdown ZFAS1 for 48 h.Actinomycin D?final concentration 5?g/m L?was added and collect samples RNA according to the time courses.ZFAS1 and NOP58 expression were detected by qPCR.Draw the degradation curve according to the RNA expression of ZFAS1and NOP58 at different time points and calculate the degradation half-life(t_1/2)according to the formula.The degradation rate of EIF4A3 and LAMC2 was also investigated in the SNORD12C silencing model.5.Cycloheximide translation activity SW620 cells transfected with ASO-SNORD12C and SNORD78 for 48h,cycloheximide?final concentration of 200?g/m L?was added,and then collect protein at different time points,LAMC2 and EIF4A3 protein expression were detected by Western Blot.6.RNA pull down the protein was extracted from HEK-293T cells transfected with shZFAS1#1.The agarose beads were incubated with ZFAS1-wild,ZFAS1-mut or ZFAS1-antisense probes for 4-5h,then the beads-probe complex was incubated with the protein overnight,and western blot was used to detect NOP58 expression.7.RTL-P to detect 2'-O-methylation Designing specific RT primers,upstream and downstream primers of methylation sites,reverse transcription in low dNTP and PCR,5?l of sample was taken for agarose gel electrophoresis.8.Nude mice experiment Healthy BALB/C nude mice were subcutaneously inoculated into the right upper axilla with 5×10⁶ cells.The tumor size and weight were measured in half of the mice,and the survival curves were drawn according to the other half.9.In situ hybridization and immunohistochemistry In situ hybridization?ISH?was used to detect the expression of ZFAS1;Immunohistochemistry?IHC?was used to detect the expression of NOP58 in colorectal cancer tissues.10.Statistical Analysis Data were analyzed with SPSS 19.0 statistical software?SPSS Inc,Chicago,USA?,expressed as mean±standard deviation?`x±SD?.P<0.05 indicates statistical significance.One-Way ANOVA and Independent t test were used for comparisonbetween groups.Pearson?2 and Fisher's test were used to analyze the correlations betweenZFAS1 expression and clinicopathological parameters.Kaplan-Meier plot was used to draw the survival curves.Results:1.ZFAS1 targets the recruitment of NOP58 to promote the biological characteristics of colorectal cancer cells.?1?Differential expression the analysis of LncRNA-mRNA microarray and TCGA database showed that ZFAS1 and NOP58 were highly expressed.The correlation coefficient of ZFAS1 with NOP58 was the highest?R²=0.5283,P<0.0001?.The expressions of ZFAS1 and NOP58 in HCT116 and SW620 cells were also up-regulated?P<0.01,P<0.001,P<0.0001?.?2?Cell phenotype Knockdown ZFAS1 significantly decreased the proliferation viability of HCT116 and SW620 cells?P<0.001?.On the contrary,the cell viability of overexpressing ZFAS1 was significantly increased?P<0.0001?.Transwell assay showed that the migration ability of CRC cells was declined after silencing ZFAS1,and the migration ability was significantly increased after overexpression of ZFAS1?P<0.05,P<0.01,P<0.001?.After silencing ZFAS1,the percentage of apoptosis of CRC cells was significantly increased?P<0.01?;while the percentage was significantly decreased after overexpressing ZFAS1?P<0.01?.The results indicate that ZFAS1 can act as an oncogene to promote CRC cell proliferation,metastasis,and inhibit CRC cell apoptosis.?3?ZFAS1 is involved in CRC cell biological functions associated with NOP58.Cell localization analysis of ZFAS1 by in situ hybridization combined with immunofluorescence showed that ZFAS1 was expressed in both nucleus and plasma,and co-localized with NOP58.The silencing or overexpression of ZFAS1 in HCT116 and SW620,the expression of NOP58 was significantly down-regulated with the silence of ZFAS1?P<0.01,P<0.001,P<0.0001?,and the expression levels of NOP58 was significantly increased after overexpression of ZFAS1?P<0.01,P<0.001,P<0.0001?.The NOP58 protein in CRC cells was detected by Western Blot and immunofluorescence,and the trend was the same as that of qPCR.mRNA stability showed that the degradation time of NOP58 m RNA was significantly shortened after silencing ZFAS1,the degradation rate was significantly accelerated and the half-life was shortened with the decrease of ZFAS1.The above results indicate that NOP58,a key protein that plays a scaffolding role during the formation of snoRNP complex,may be regulated by ZFAS1.2.ZFAS1 recruits NOP58 to regulate the molecular mechanism of SNORD12C/78-mediated rRNA 2'-O-methylation in colorectal cancer.?1?NOP58 protein was pulled down by ZFAS1 probe,the expression of the shZFAS1#1 group was significantly lower than that of the control,indicating that ZFAS1and NOP58 bind directly.?2?SNORD12C/78 co-expression with NOP58 were screened and determine by co-expression microarray.After silencing or overexpression of NOP58,SNORD12C and SNORD78 was also reduced or enhanced,and the 2'-O-methylation levels also decreased or increased,correspondingly?P<0.01,P<0.001,P<0.0001?.The expression of NOP58,SNORD12C and SNORD78 was rescued by ZFAS1,as well as the 2'-O-methylation levels,indicating that ZFAS1 regulates the recruitment of SNORD12C and SNORD78 by specifically binding NOP58.?3?SNORD12C/78 is involved in the regulation of downstream target genes.The relevant factors regulating the expression of ZFAS1 and SNORD12C/78 were mainly concentrated in the ribosome pathway by GO functional enrichment analysis.Representative target genes LAMC2 and EIF4A3 were selected to futher research.After ASO-SNORD12C and ASO-SNORD78,mRNA and protein expression of LAMC2 and EIF4A3 were significantly decreased.ZFAS1 was able to rescue the expression level after ASO-SNORD12C/78 knockdown.The mRNA stability assay found that LAMC2 and EIF4A3 decreased with silencing of SNORD12C,and the half-life was shortened more obviously.Actinomycin?CHX?was used to investigate protein translational activity after silencing SNORD12C and SNORD78.The results showed that LAMC2 and EIF4A3 were significantly degraded at about 4-6 h.?4?Nude mice were used to verify the correlation of ZFAS1 and NOP58 in vivo.The tumor volume and weight of pcDH-ZFAS1+NOP58-wild group were significantly larger than those of pcDH-ZFAS1 group.The survival time of the pcDH-ZFAS1+NOP58-wild group was significantly shorter than that of the pcDH-ZFAS1+NOP58-mut group and the pcDH-ZFAS1 group.Western bolt and qRT-PCR also showed that the expression of NOP58 was most significant in the pcDH-ZFAS1+NOP58-wild group.The above results demonstrate that ZFAS1 promotes the CRC proliferation via NOP58 in nude mice.Conclusions:1.ZFAS1 is abnormally highly expressed in colorectal cancer cells and tissues,and can be used as an oncogene to promote cell proliferation and migration and inhibit apoptosis.The analysis of the correlation between ZFAS1 and clinicopathological parameters and prognosis showed that when ZFAS1 is overexpressed,the disease-free survival and overall survival of patients are shortened,and the prognosis is poor.2.Comprehensive analysis of colorectal cancer cell lines,tissues,and large sample databases showed that the key protein of snoRNP complex,NOP58 was significantly positively correlated with ZFAS1 expression.ZFAS1 and NOP58 bind directly,and NOP58 is regulated by ZFAS1.It is also confirmed that ZFAS1 can promote cell phenotypic changes such as colorectal cancer proliferation by specifically binding NOP58in vivo and in vitro.3.NOP58 accelerates the recruitment of SNORD12C and SNORD78 and promotes the level of 2'-O-methylation of the rRNA G3878 and G4593 sites.4.ZFAS1 promotes the 2?-O-methylation level of rRNA G3878 and G4593 by SNORD12C and SNORD78 through targeted binding to NOP58,improves the translation activity of proteins such as LAMC2 and EIF4A3,and finally promotes the proliferation of colorectal cancer cells.The new molecular mechanism of ZFAS1 in colorectal cancer proliferation was clarified at the level of epigenetic modification.Reversing the level of rRNA 2'-O-methylation may become a new treatment method for colorectal cancer.