The Role And Mechanism Of SNORA71C In Regulating Biological Characteristics Of Colorectal Cancer Cells Through CHD3/TBP/SULF1 Pathway

Objective: Colorectal cancer(CRC)is a common malignancy worldwide,and its morbidity and mortality remain increasing year by year.According to the statistics of the National Cancer Center in 2022,CRC ranks second in morbidity and fourth in mortality among cancers in China,with 408,000 new cases and 196,000 deaths.In recent years,with the development of multiple department treatment(MDT),the treatment effect of CRC has improved,but the five-year survival rate of CRC with locally advanced stage and distant metastasis was 53% and 14%,respectively.Tumor invasion and metastasis is the primary cause of poor prognosis of CRC.Conventional treatment methods cannot improve the prognosis of patients with advanced tumors.Therefore,further elucidating the molecular mechanism of CRC invasion and metastasis and seeking new therapeutic targets are of special significance for improving the treatment efficacy of CRC.Small nucleolarRNAs(snoRNAs)are a class of small non-codingRNAs(ncRNAs)localized in the nucleus of eukaryotic cells,with a length of 60-300 nucleotides,and are mainly classified into box C/D SNORD and box H/ACA SNORA according to their structure and function.The classical biological function of snoRNAs is to participate in the post-transcriptional modification process of ribosomal RNA(r RNA).snoRNAs are aberrantly expressed in malignant tumors and can participate in the biological processes such as tumorigenesis,progression,metastasis and drug resistance of tumors.For example,SNORA42,SNORD78 and SNORA23 have been reported to be aberrantly expressed in different types of tumor tissue,cells and patients body fluids.Therefore,further investigation of the relationship between snoRNAs and CRC progression and metastasis may provide new ideas for the treatment of advanced CRC.Sulfatase 1(SULF1)is a cell surface enzyme that selectively removes the 6-O-sulfate group of heparan sulfate proteoglycans(HSPGs)from the cell surface to regulate cell signaling,thereby affecting a variety of physiological and pathological processes.In CRC,bioinformatic analysis and cell function assays have confirmed that SULF1 can affect the proliferation of tumor cell,migration and invasion,and its high expression also leads to alteration of the tumor immune microenvironment.With the deepening of research,it is also found that SULF1 can be acted as a target gene together with ncRNAs and transcription factors to build a regulatory network,which affects malignant tumor progression and prognosis.These results provide a new direction and idea to explore the regulatory role between ncRNAs and SULF1.Based on CRC cell lines,this study took SNORA71C as the entry to elucidate the effect of SNORA71C regulation of SULF1 on the biological behavior of colorectal cancer cells,and further identified the molecular mechanism that SNORA71C may affect the transcription of SULF1 by transcription factor TBP through combining with CHD3,and then regulate the biological characteristics of colorectal cancer.Through this study,we discovered and confirmed the mechanism of SNORA71C in regulating the biological characteristics of CRC,which is expected to provide new ideas for elucidating the mechanism of snoRNAs in malignant tumor and provide relevant theoretical support for reversing the progression of cancer patients and discovering new clinical diagnosis and treatment targets.Method:1.Screening,identification and bioinformatics analysis of sno RNA: the differentially expressed SNORA71C and SULF1 in CRC and paired para-carcinoma tissues were screened by high-throughput sequencing.The expression level of SNORA71C in CRC cells and tissue were verified by q RT-PCR and in situ hybridization assays.The effects of SNORA71C on prognosis of patients with colorectal cancer were analyzed through TCGA database.We utilized the clinicopathologic information of patients with tissue microarry,Kaplan-Meier method was used to analyze the influence of SNORA71C expression level on patients prognosis.Further,Fisher test,Logistic regression analysis and Cox regression model were used to elucidate the relationship between SNORA71C and clinicopathologic parameters in patients with colorectal cancer.Finally,we applied co-expression analysis of sno RNA-m RNA and transcriptome sequencing of sno RNA,SULF1 was found to be significantly correlated with SNORA71C.2.SNORA71C regulates SULF1 mediated TGF-β1/Smad pathway: antisense oligonucleotides(ASO)of SNORA71C were transfected into the screened CRC cell lines HCT116 and SW480 to construct SNORA71C silencing model.SNORA71C overexpression lentivirus was transfected into the same cell line to construct SNORA71C overexpression model.The transfection efficiency was detected by q RT-PCR assay.The alteration of SULF1 protein level in SNORA71C overexpressed cells were detected by Western blot assay.The protein levels of SULF1 related TGF-β1/Smad pathway were further detected by Western blot assay.3.Effect of SNORA71C on the malignant biological behavior associated with CRC cells:In CRC cell lines HCT116 and SW480,CCK8 and colony formation assays were performed to detect the changes of cell proliferation ability after intervention of SNORA71C.The proportion of apoptosis was detected by flow cytometry.Wound-healing and Transwell assays were performed to detect the changes of cell migration and invasion abilities after intervention of SNORA71C.The protein levels of migration and invasion indicators E-cadherin,Snail and Vimentin were detected through Western bolt assay.4.SNORA71C regulates the expression of SULF1 through CHD3/TBP/SULF1 pathway:Specific molecular probe was designed and applied to enrich protein which can bind to SNORA71C in HCT116 cells.Further mass spectrometry was performed to identify the protein type.The combination of SNORA71C and CHD3 was verified by Ch IRP and RIP assays.Based on STRING,PROMO and JASPAR databases,transcription factors that could interact with CHD3 were screened out.Co-IP and Western blot were used to detect the combination of TBP and CHD3.The relationship between transcription factor TBP and target gene SULF1 was detected through Ch IP and dual-luciferase assays.5.Design and construction of recovery assay and animal model: We first constructed a cell model with dual intervention of SNORA71C and SULF1.Then we detected the changes in cell proliferation,migration and invasion ability through CCK8,Colony formation and Transwell assays.The protein levels of migration and invasion indicators were detected by Western bolt assay.Finally,a nude mouse transplanted tumor model was established to verify the molecular mechanism of SNORA71C regulating the biological characteristics of colorectal cancer through CHD3/TBP/SULF1 pathway in vivo.Results:1.SNORA71C and SULF1 were highly expressed in colorectal cancer and mediate poor prognosis.There was a positive correlation between them.(1)SNORA71C is highly expressed in colorectal cancer and correlated with SULF1: Based on the differential expression profile of high-throughput sno RNA-m RNA tissue microarry in colorectal cancer and paired adjacent tissue,q RT-PCR and in situ hybridization assays were performed to screen and verify SNORA71C with the most obvious differential expression in colorectal cancer cells and tissue(p < 0.01,p < 0.0001).Further FISH assay suggested that SNORA71C was significantly higher expressed in colorectal cancer cells compared with human normal intestinal epithelial cells.(2)SNORA71C was associated with poor prognosis in colorectal cancer patients: Based on TCGA database,the progression free interval was significantly shortened in CRC patients with high expression of SNORA71C(p < 0.05,p < 0.01).There was a linear positive correlation between SNORA71C expression level and abnormal copy number of colorectal cancer(p < 0.001).Further analysis based on clinicopathological information of patients with tissue microarry indicated that the overall survival of CRC patients with high expression of SNORA71C was shorter(p < 0.01).Subsequent Fisher test and Logistic regression analysis suggested that patients with high expression of SNORA71C had lower differentiation(p < 0.05)and increased tumor invasion depth(p < 0.05).Multivariate Cox analysis showed that high expression of SNORA71C was an independent prognostic factor in CRC patients(p < 0.05).(3)SULF1 was highly expressed in colorectal cancer and had a positive correlation with SNORA71C expression: Based on sno RNA-m RNA co-expression analysis,SULF1 was significantly highly expressed in colorectal cancer,and there is a significant positive correlation between SNORA71C and SULF1(R = 0.102,p < 0.01).Data analysis suggested that the expression of SULF1 was elevated in colorectal cancer tissue(p < 0.01)and could lead to poor prognosis in patients as well(p < 0.01).We obtained SULF1 through the intersection of transcription sequencing differential genes and m RNA co-expression profiles.This suggested that SNORA71C and SULF1 might have a regulatory relationship.2.SNORA71C can affect the biological behavior of CRC cells by regulating SULF1.(1)SNORA71C can up-regulated SULF1 and then activated TGF-β1/Smad pathway:Firstly,HCT116 and SW480 cell models were constructed to interfere with the expression of SNORA71C.SULF1 protein level was significantly elevated through Western blot assay(p < 0.01).Further Western blot results indicated that high expression of SULF1 could induce up-regulation of TGF-β1(p < 0.01),Psmad2(p < 0.01)and Psmad3(p < 0.01)protein levels in TGF-β1/Smad pathway.(2)The effects of SNORA71C on the biological behavior of colorectal cancer cells: The proliferation and colony formation abilities of HCT116 and SW480 cells were significantly decreased after silencing SNORA71C(p < 0.01);The proportion of apoptosis was significantly increased(p < 0.01);Cell migration ability(p < 0.001)and invasion ability(p < 0.01)were significantly decreased.The proliferation and colony formation abilities of HCT116 and SW480 cells were significantly increased after overexpression SNORA71C(p < 0.01),while the percentage of apoptosis was significantly decreased(p < 0.01).Cell migration ability(p < 0.01)and invasion ability(p < 0.001)were significantly increased.Western blot assay was perfromed to detect migration and invasion related indicators.The results indicated that the expression of E-cadherin(p < 0.05)decreased,Snail(p < 0.05)and Vimentin(p < 0.01)increased after overexpression of SNORA71C.3.The target and specific mode of SNORA71C in regulating SULF1.(1)SNORA71C regulated the expression of SULF1 by targeting CHD3: CHD3 directly bind to SNORA71C was screened out according to mass spectrometry analysis results.The results of immunofluorescence co-localization assay also suggested that SNORA71C and CHD3 were co-expressed in the nucleus of HCT116 and SW480 cells.The combination between SNORA71C and CHD3 was further verified through RIP and Ch IRP assays.(2)CHD3 can interact with TBP to induce the activation of SULF1 transcription: The SULF1 potential transcription factor TBP that might interact with CHD3 were screened out based on PROMO and STRING databases.Co-IP and Western blot assays confirmed the combination between CHD3 and TBP.In SNORA71C overexpression cell models,we found that the binding of CHD3 to TBP was significantly decreased(p < 0.01),and the expression of free TBP that plays the transcriptional function in the nucleus was significantly increased(p < 0.01).Furthermore,we silenced TBP in HCT116 and SW480cells(p < 0.05),then the expression level of SULF1 decreased significantly(p < 0.01),suggesting that SULF1 might be controlled by TBP.Ch IP,agarose gel electrophoresis and dual-luciferase assays suggested that TBP could specifically bind to the promoter region of SULF1(p < 0.01)and exert transcriptional activation function(p < 0.01).4.Rescue assay confirmed SNORA71C can regulate the biological characteristics of colorectal cancer cells through CHD3/TBP/SULF1 pathway.(1)In vitro cell assays verified the changes of TGF-β1/Smad pathway related indicators and cell phenotypes after simultaneous intervention of SNORA71C and SULF1: Dual intervention cell models of SNORA71C and SULF1 were successfully constructed in HCT116 and SW480 cells.The results of Western blot assay suggested that the activation of TGF-β1/Smad pathway related indicators caused by overexpression of SNORA71C could be reversed by silencing SULF1.Subsequent results of CCK8 and colony formation assays suggested that the increase of cell proliferation ability induced by overexpression of SNORA71C could be reversed by silencing SULF1.Transwell assay results also suggested that the increase of cell migration and invasion abilities induced by overexpression of SNORA71C could be reversed by silencing SULF1.Further Western blot assay was performed to detect the changes in the protein levels of migration and invasion related indicators in the dual-intervention cell model.The results indicated that the related indicators had corresponding trends with the groups.(2)Effects of dual intervention of SNORA71C and SULF1 on tumor growth,metabolism and related pathway indicators in nude mouse tumor transplantation model.After subcutaneous injection of SW480 cells to build a nude mouse transplantation tumor model,we found that overexpression of SNORA71C could promote tumor metabolism and growth.The above effects could also be reversed by silencing SULF1.In transplantation tumors overexpressed with SNORA71C,immunohistochemistry(IHC)results indicated that Psamd2/3 was increased in TGF-β1/Smad pathway while E-cadherin,an indicator related to migration and invasion,was reduced.Vimentin expression is increased.The above trend can also be reversed by silencing SULF1.The changes of various indicators in the animal model were consistent with the results in vitro.These results together verified that SNORA71C can regulate the biological characteristics of colorectal cancer through the CHD3/TBP/SULF1 pathway.Conclusions:1.SNORA71C was aberrantly highly expressed in colorectal cancer and positively correlated with the expression of SULF1;2.In CRC patients with high expression of SNORA71C,progression free interval and overall survival were shortened,SNORA71C could lead to poor prognosis as an independent prognostic factor;3.SNORA71C can positively regulate SULF1 to activate the classic TGF-β1/Smad pathway and promote the proliferation,migration and invasion of colorectal cancer cells;4.SNORA71C can weaken the interaction between CHD3 and transcription factor TBP by targeting CHD3 which result in promoting the transcriptional of SULF1 through elevating the level of free TBP.That is,SNORA71C can activate TGF-β1/Smad pathway through CHD3/TBP/SULF1 pathway and promote the alteration of biological characteristics of colorectal cancer cells.