The Regulatory Effect And Mechanism Of SNORD18B/18C On The Growth And Radiosensitivity Of Colon Cancer

Colon cancer is one of the most common malignant tumors worldwide.In recent years,due to the improvement of living standards and lifestyle changes,the incidence and mortality of colorectal cancer have been elevated steadily in China,and there is a younger trend at disease onset.Therefore,colorectal cancer,being a complex multifactorial disease among gastrointestinal tract cancer,is threatening the health of Chinese people.Because colonoscopy screening is not pervasive and most of the cases are asymptomatic,the patients usually diagnosed with moderately advanced/advanced colon cancer had a poor prognosis.Surgery,chemotherapy and radiotherapy are the main methods of colon cancer treatment at present.Although therapeutic modalities have been optimized,there is still room for improvement of the prognosis in patients with colon cancer.So,the search for new prognostic markers and for new molecular mechanism of radiosensitivity will help further improve the prognosis of colon cancer patients.Small nucleolar RNAs（snoRNAs）have been recognized as important regulatory molecules of biological processes in various diseases,particularly oncogenesis.In recent decades,reports on the role of snoRNAs in tumors have been increasing,exploration of the biological function and mechanism of snoRNAs in tumors will provide novel prognostic markers and potential therapeutic targets for cancer patients.Radiotherapy is one of the treatment options for patients with colon cancer.However,some patients are not sensitive or even completely resistant to radiation,which is also an important factor restricting further improvement of prognosis for patients with colon cancer.At present,the research on the role of snoRNA in tumor radiosensitivity is still in the blank stage,and the study on the participation of snoRNA in regulating the radiosensitivity of tumor cells has not been reported so far.Therefore,further exploration of the mechanism behind this field is full warranted.With the combination of the results of snoRNA microarray and our previous research which has screened out human and mouse homologous snoRNA with potential radiation response through mouse model,we finally identified two snoRNAs（SNORD18B and SNORD18C）that may have important regulatory functions in colon cancer.Through clinical database investigations,in vitro and in vivo functional experiments,high-throughput sequencing and bioinformatics analysis,we have confirmed its regulatory effect on the growth and radiosensitivity of colon cancer cells at the different levels.The underlying mechanism of its biological function was also further explored,which might provide a novel biomarker for the diagnosis,prognosis and radiosensitivity in patients with colon cancer,and also might provide a novel potential target for the treatment of colon cancer.The experimental study was carried out in four parts to investigate the regulatory effect and mechanism of SNORD18B/18C on the growth and radiosensitivity of colon cancer.Part I:The expression profile of SNORD18B/18C in colon cancer and its clinical significanceObjective:To screen out the candidate snoRNAs for diagnosis,prognosis and radiosensitivity assessment of colon cancer and explore its expression profile and clinical significance in colon cancer.Methods:The potential radiation response of human and mouse homologous gene snord18b and snord18c was verified by qPCR.Twenty-five pairs of matched colon cancer and adjacent normal mucosa tissues and different colon cancer cell lines were obtained to evaluate the expression of SNORD18B/18C via qPCR.ROC curves were used to analyze the diagnostic value of SNORD18B and SNORD18C in colon cancer.We collected colon cancer patients’data from the TCGA database to evaluate the relationship between SNORD18B/18C expression and clinicopathological parameters.The association between SNORD18B/18C expression and clinicopathological parameters was analyzed by using a chi-square test.Spearman’s correlation analysis was used to evaluate the correlation between expression of SNORD18B and SNORD18C.Survival curves were plotted by using the Kaplan-Meier method and the log-rank tests was applied to analyze the different survival groups using the SNORD18B/18C expression median value as the cutoff.The multivariate Cox proportional hazard models were used to evaluate the relationship between SNORD18B/18C expression and survival.Results:Snord18b and snord18c were significantly up-regulated in the irradiated group when compared with the non-irradiated group.When normalized to FHC,the expression of SNORD18B and SNORD18C were shown to be significantly increased in all 3 colon cancer cell lines（HCT116,SW620 and HT2929）.SNORD18B and SNORD18C were significantly overexpressed in tumor tissues compared with normal mucosa tissues,which was consistent with the cell line results.Furthermore,the receiver operating characteristic（ROC）curve analysis which indicated SNORD18B and SNORD18C expression could discriminate colon cancer from normal mucosa tissues suggested that SNORD18B and SNORD18C could be used in colon cancer diagnosis.SNORD18B expression was significantly associated with age,lymphatic invasion,venous invasion and history of colon polyps,while SNORD18C expression was significantly associated lymphatic invasion and history of colon polyps.Spearman’s correlation analysis showed that the expression of SNORD18B and SNORD18C was significantly associated.The results of Kaplan-Meier analysis revealed that patients with high SNORD18B or SNORD18C expression had a worse overall survival,and those with both high expression of SNORD18B and SNORD18C had the worst prognosis.The multivariate analysis indicated that only age,TNM stage,SNORD18B high expression,SNORD18C high expression and both SNORD18B and SNORD18C high expression were the independent prognostic factors for colon cancer patients’overall survival.Conclusions:（1）Ionizing radiation could induce the overexpression of snord18b and snord18c in peripheral blood lymphocytes of mice,suggesting that they may have potential correlation with genomic instability,immune response or radiation response;（2）SNORD18B and SNORD18C were overexpressed in colon cancer tissues and cell lines,and they could be used as diagnostic biomarkers in colon cancer;（3）Patients with high SNORD18B or SNORD18C expression had a worse overall survival,SNORD18B and SNORD18C were the independent prognostic factors for overall survival in patients with colon cancer,which suggesting that they could be considered as prognostic biomarkers in colon cancer.Part II:The regulatory effect of SNORD18B/18C on proliferation,invasion and migration of colon cancer cellsObjective:To investigate the regulatory effect of SNORD18B/18C on proliferation,invasion and migration of colon cancer cells.Methods:Small interference RNA（siRNA）and CRISPR-Cas9techniques were used to establish the SNORD18B/18C knock-down and knock-out cell model,respectively.Lentiviral vector was used to establish the SNORD18B and SNORD18C overexpression cell model.The SNORD18B/18C down or up-regulated effect of above experiments was verified by qPCR.CCK-8 and colony formation experiments were performed to investigate the cell proliferation and colony formation.Transwell and wound healing assays were used to evaluate the migration and invasion.Nude mice experiment was used to confirm the effect of SNORD18B and SNORD18C on tumorigenicity in vivo.Results:QPCR verified that the SNORD18B/18C down and up-regulated cell lines were constructed successfully.CCK-8 and colony formation experiments showed that SNORD18B/18C knock-down or knock-out colon cancer cell lines exhibited significantly decrease in cell growth and clonogenicity,while overexpressing SNORD18B or SNORD18C exhibited increased cell proliferation and colony formation ability.The results of nude mice experiment revealed that knockdown SNORD18B or SNORD18C suppressed the tumorigenicity in vivo,while overexpressing SNORD18B or SNORD18C promoted the umorigenicity in vivo.Transwell and wound healing assays demonstrated that SNORD18B or SNORD18C promoted the migration and invasion,and knock-down SNORD18B/18C inhibited the migration and invasion of colon cancer cells.Conclusions:（1）SNORD18B and SNORD18C could positively regulate the proliferation and clonogenicity of colon cancer cells;（2）SNORD18B and SNORD18C could positively regulate the invasion and migration of colon cancer cells;（3）SNORD18B and SNORD18C could positively regulate the tumorigenicity in vivo.Part III:The regulatory effect of SNORD18B/18C on radiosensitivity of colon cancer cellsObjective:To investigate the regulatory effect of SNORD18B/18C on radiosensitivity of colon cancer cells.Methods:Colony formation experiment combined with multi-target single hit model analysis was used to evaluate the radiosensitivity of colon cancer after SNORD18B/18C knock-out or overexpression.Apoptosis and cell cycle detection kit were used to detect the apoptosis and cell cycle,respectively.To evaluate the effect of SNORD18B or SNORD18C on apoptosis induced by irradiation or hydrogen peroxide,apoptosis rate of stable overexpression cell lines were detected after 10Gy radiation or H₂O₂（400μM for 8h）treatment.Western blot was used to investigate the effects of SNORD18B/18C on the major proteins in non-homologous end joining（NHEJ）pathway,cell cycle related proteins and DNA double strand break markerγ-H2AX protein.Furthermore,the NHEJ repair efficiency of SNORD18B and SNORD18C knock-out cell lines were detected via NHEJ-GFP fluorescence report system.Results:The results of multi-target single hit model analysis showed that the mean lethal dose,quasi threshold dose and survival fraction of SNORD18B or SNORD18C knock-out cell lines were lower than those of the control group,while the mean lethal dose,quasi threshold dose and survival fraction of stable overexpression cell lines were higher than those of the control group.In other words,SNORD18B and SNORD18C could negatively regulate the radiosensitivity of colon cancer cells.SNORD18B/18C inhibition resulted in a marked increase in the percentage of apoptotic cells,while overexpressing SNORD18B or SNORD18C inhibited the apoptosis of colon cancer cells.After treated with 10Gy radiation or H₂O₂（400μM for 8h）treatment,stable SNORD18B and SNORD18C overexpression cell lines exhibited significantly decrease of apoptosis rate when compared with the control group.Western blot data showed that knock-down SNORD18B/18C could increase the expression level ofγ-H2AX and suppress the expression level of DNA-PKcs,Lig4 and Bcl-2.SNORD18B and SNORD18C knock-out cell lines exhibited a significantly decrease in NHEJ repair efficiency.Knock-down SNORD18B/18C induced significant G0/G1-phase arrest in colon cancer cells,while overexpressing SNORD18B or SNORD18C promoted cell cycle from G0/G1-phase to S and G2/M-phase.Western blot data of cell cycle related proteins showed that knock-down SNORD18B/18C could induce G0/G1-phase arrest by activating the phosphorylation of ATM and CHK2,suppressing the phosphorylation of CDK2 and inhibiting the expression of Cycline E.Conclusions:（1）SNORD18B and SNORD18C could negatively regulate radiosensitivity of colon cancer cells;（2）SNORD18B and SNORD18C could negatively regulate the apoptosis of colon cancer cells to reduce the apoptosis induced by ionizing radiation;（3）SNORD18B and SNORD18C involved in the expression of major proteins in the NHEJ repair pathway could affect the DNA damage repair of colon cancer cells by regulating the efficiency of NHEJ repair;（4）SNORD18B and SNORD18C were involved in the regulation of cell cycle,and knock-down SNORD18B/18C induced G0/G1-phase arrest of colon cancer cells.Part IV:The molecular mechanism of SNORD18B/18C regulating the growth and radiosensitivity of colon cancer cellsObjective:To explore the molecular mechanism of SNORD18B/18C regulating the growth and radiosensitivity of colon cancer cells.Methods:Nuclear/Cytosol Fractionation Kit was used to analyze the subcellular localization of SNORD18B and SNORD18C.Western blot and qPCR were used to explore the influence of SNORD18B/18C on RPL4 expression at protein and mRNA level,respectively.RNA-seq and bioinformatics analysis were used to screen out the potential downstream signal pathway of SNORD18B/18C.Combining the signal pathway enrichment analysis results of differential mRNA expression of SNORD18B and SNORD18C in TCGA database,we screened out the most reliable potential downstream signal pathway.Finally,qPCR and western blot were used to verify the expression of signal pathway associated proteins.Results:SNORD18B and SNORD18C were mainly located in the nuclear of colon cancer cells.Knock-down SNORD18B/18C did not affect the expression of RPL4 at mRNA and protein levels.GO-enriched analysis showed that the differential expression gene mainly involved in the biological process of transcription regulation,multicellular organism,signal transduction and ion transport;The differential expression gene mainly located in the membrane and nucleus,and the major molecular function were protein binding,metal ion binding,DNA binding and DNA-binding transcription factor activity.After synthesizing the KEGG Pathway enrichment results of the RNA-seq and TCGA database,we screened out PI3K-AKT-CREB signal pathway as the most reliable downstream signal pathway of SNORD18B/18C.Knock-down SNORD18B/18C suppressed the expression of AKT3 and CREB3L3 at mRNA level.The result from western blot data that knock-down SNORD18B/18C inhibited the expression of PI3K,p-PI3K,AKT,p-AKT,CREB and p-CREB indicated that SNORD18B/18C might regulate the growth and radiosensitivity of colon cancer cells through PI3K-AKT-CREB signal pathway.Conclusions:（1）SNORD18B and SNORD18C were mainly located in the nuclear of colon cancer cells;（2）SNORD18B/18C did not affect the expression of RPL4 at mRNA and protein levels;（3）Knock-down SNORD18B/18C inhibited the expression of PI3K,p-PI3K,AKT,p-AKT,CREB and p-CREB;（4）SNORD18B and SNORD18C might regulate the growth and radiosensitivity of colon cancer cells through PI3K-AKT-CREB signal pathway.Conclusions:（1）SNORD18B and SNORD18C were overexpressed in colon cancer tissues,and they could be used as diagnostic biomarkers for colon cancer;（2）Patients with high SNORD18B or SNORD18C expression had a worse overall survival and they were the independent prognostic factors for colon cancer patients’overall survival,and these results suggested that they could be used as prognostic biomarkers in colon cancer;（3）SNORD18B and SNORD18C could positively regulate the growth,migration and invasion of colon cancer cells;（4）SNORD18B and SNORD18C could negatively regulate radiosensitivity of colon cancer cells;（5）SNORD18B and SNORD18C might regulate the growth and radiosensitivity of colon cancer cells through PI3K-AKT-CREB signal pathway.