Role And Mechanism Of Small Nucleolar RNA SNORA28 Activating STAT3 In The Growth Of Colon Cancer Cells

Colon cancer is a common and highly malignant gastrointestinal tumor.The incidence rate ranks first in malignant tumors of digestive system.At present,the main treatment of colon cancer is surgery,combined with radiotherapy,chemotherapy,immunotherapy,molecular targeted therapy and traditional Chinese medicine treatment and other means of comprehensive treatment.Because the early symptoms of colon cancer are not obvious,some patients lack early screening,so that when colon cancer patients are diagnosed,the disease may have developed to the middle and late stages,which seriously threatens the patient’s life.Despite advances in the treatment of colon cancer in recent years,the prognosis of patients is still poor,and metastatic recurrence after surgery is the main cause of death.Therefore,clarifying the occurrence and development of colon cancer and exploring new molecular biomarkers of colon cancer are of great significance for the early diagnosis and later treatment of colon cancer.Sno RNA is a class of small non-coding RNA that widely exists in the nucleolus of eukaryotic cells,and it exists stably in urine,sputum,plasma and blood,which means that sno RNA has potential value as a molecular biomarker for early disease diagnosis and prognosis.Objective: In this study,the differentially expressed sno RNA molecules in colon cancer tissue and adjacent tissue were screened by nr Star^TM Human sno RNA PCR microarray.Further analysis was made on the screened sno RNA molecules in clinical samples and cell lines of colon cancer to explore its biological characteristics and mechanism of action in colon cancer.It provides a new perspective for the early diagnosis and late treatment of colon cancer.Methods:1.We collected 27 pairs of cancer and adjacent tissue specimens from patients diagnosed with colon cancer in the Colorectal Surgery Department of Liaoning Cancer Hospital,and collected clinicopathological information of the patients for later research.2.We randomly screened the cancer and adjacent tissues of 3 patients who had been diagnosed with colon cancer.Nr Star ^TM Human sno RNA PCR chip technology and bioinformatics method were used to screen out sno RNA differentially expressed in colon cancer,which was verified by real-time q PCR.Finally,SNORA28 was selected as the sno RNA molecule in this study.Meanwhile,the related database of The Cancer Genome Atlas（TCGA）was further analyzed to determine the expression of SNORA28 in other tumor tissues.3.The expression of SNORA28 in 27 pairs of colon cancer and adjacent tissues,as well as in colon cancer cell lines and normal colon epithelial cells was verified by real-time q PCR.At the same time,according to the further analysis of the colon cancer data information in the TCGA database,there were a total of 452 colon cancer patients,the incomplete patient data were excluded,and finally the data information of 277 colon cancer patients were included in this study.The relationship between SNORA28 expression and clinicopathological features,prognosis and survival of colon cancer patients was analyzed in detail to explore its potential value for clinical research.4.Two colon cancer cell lines,HCT116 and HT29,were selected for subsequent cell phenotyping experiments.SNORA28 was overexpressed and knocked down by infection with lentivirus and transfection with ASO knockdown sequence.CCK8 assay,clone formation assay,cell cycle and apoptosis assay were performed to detect the effects of SNORA28 on proliferation ability,clone formation,migration and invasion,cell cycle and apoptosis of colon cancer cells.5.HCT116 cells were infected with lentivirus and transfected with ASO knockdown sequence for SNORA28 overexpression and knockdown.The overexpression and knockdown effects were verified by real-time q PCR.Transcriptome sequencing was performed on samples overexpressing and knocking down SNORA28,respectively.The differentially expressed genes,GO enrichment and signal pathway enrichment analysis were further verified to determine the molecular mechanism of SNORA28 regulating colon cancer cell growth.Results:1.The nr Star^TM Human sno RNA PCR chip was used to detect the cancer and adjacent tissues of colon cancer patients.A total of 143 sno RNAs were found to be differentially expressed in colon cancer tissues,of which 75 were up-regulated and 67 were down-regulated.2.The tumor data in the TCGA database was analyzed,and it was found that SNORA28 was highly expressed in a variety of tumors.3.Expanded samples to verify that SNORA28 is highly expressed in colon cancer tissues and colon cancer cell lines4.The relationship between the expression level of SNORA28 and clinicopathological parameters was evaluated by ROC curve,Kaplan-Meier analysis and Cox regression analysis,indicating that the expression of SNORA28 is associated with poor prognosis.5.Overexpression of SNORA28 can promote colon cancer cell proliferation,clone formation and tumorigenicity in nude mice.Knockdown of SNORA28 can significantly inhibit colon cancer cell proliferation,clone formation and induce colon cancer cell apoptosis.6.Transcriptome sequencing analysis of HCT116 cells overexpressing and knocking down SNORA28.Differential genes were enriched to the JAK-STAT signaling pathway and overexpression of SNORA28 was found to activate the JAK-STAT signaling pathway.Conclusion:1.SNORA28 is highly expressed in colon cancer tissues as well as colon cancer cell lines.The expression of SNORA28 is associated with lymphatic invasion,history of colon polyps,and poor prognosis,and can be used as a potential biomarker for colon cancer diagnosis and prognosis.2.The increased expression of SNORA28 can promote colon cancer cell proliferation,clone formation and tumorigenicity in nude mice,and vice versa.SNORA28 can activate JAK-STAT pathway by transcriptome sequencing and validation analysis.Compared with normal colon epithelial cells,both SNORA28 and STAT3 proteins were highly expressed in colon cancer cells,suggesting that SNORA28 may promote the growth of colon cancer cells by activating the JAK-STAT signaling pathway.