The Function And Mechanism Of LncRNA CASC9 In Nasopharyngeal Carcinoma

Backgrounds and objectives:Nasopharyngeal carcinoma is a major kind of head and neck epithelial carcinoma.The incidence and mortality of NPC in China in 2020 was 3.00 and 1.60 per 100,000 people respectively,constituting approximately46.9% of new worldwide cases and 43.0% of dead cases.Because of its insidious onset,special anatomical location and aggressive malignancy,the diagnosis of this disease was usually delayed rendening treatment difficult.Although there are chemotherapy,radiotherapy and targeted therapy,the5-year survival rate of patients is only about 40%.Therefore,NPC has become one of the major public health issue of common concern to the community today.According to the patient’s individual genetic background combined with the characteristics of tumor biological behavior,formulating individualized treatment strategies has become an important direction for the development of current nasopharyngeal cancer treatment.Researches on the mechanism of NPC can help us to find novel effective targets for diagnosis and treatment.The occurrence and development of tumors are regulated by tumor-driving signal pathways,and the activation of related tumor-driving signal pathways is the result of continuous disorder of gene expression.Cells maintain their normal biological behavior through the orderly expression of genes,and RNA level regulation,epigenetic regulation,and protein level regulation are the keys to mediating the normal and orderly expression of genes.In recent years,RNA level regulation has been confirmed to be involved in the process of malignant transformation of cells and become a hotspot of the oncology researches.Due to the inability to encode,long non-coding RNA has not received much attention before.Along with the researches on human gene expression and regulation going deep,increasing evidences show that Lnc RNAs play a vital role in cellular physiological processes such as chromatin structure remodeling,splicing editing of transcription products,m RNA stability,and multidirectional differentiation of stem cells through the interaction with DNA,RNA,and protein molecules.Lnc RNA CASC9(cancer susceptibility candidate 9)was reported in esophageal cancer for the first time in 2015,and was later confirmed to be related to the occurrence and development of tumors in malignant tumors such as gastric cancer and breast cancer.Up to now,CASC9-related researches on nasopharyngeal carcinoma is still very limited.Previous studies have found that CASC9 highly expressed in NPC and is correlated with the poor prognosis.The molecular mechanism of CASC9 in the occurrence and development of nasopharyngeal carcinoma need to be further studied.Our study aims to investigate the relationship between CASC9 and the prognosis of patients with NPC and to study its role in progression and underlying mechanism.This subject includes the following four parts: The frist part: Bioinformatics analysis of differentially expressed genes and CASC9 in nasopharyngeal carcinoma;The second part: Expression level and clinical significance of CASC9 in nasopharyngeal carcinoma;The third part: The functional research of CASC9 in nasopharyngeal carcinoma;The fourth part: Mechanism of CASC9 gene regulation of the occurrence and development of nasopharyngeal carcinoma.The Frist Part: Bioinformatics Analysis of Differentially ExpressedGenes and CASC9 in Nasopharyngeal CarcinomaMethod1.Gene expression data and clinical information were downloaded from The Cancer Genome Atlas(TCGA)official website,R language software was used to screen the differentially expressed Lnc RNA in head and neck squamous cell carcinoma.2.Regression analysis was used to screen the DELnc RNA related to the prognosis of patients with head and neck squamous cell carcinoma.3.The online tool GEPIA was used to analyze the expression of CASC9 in head and neck squamous cell carcinoma and its correlation with prognosis.4.Raw data of NPC RNA-Seq datasets were downloaded from the Sequence Read Archive Database,R language software was used to screen the DELnc RNA.Results1.889 Lnc RNAs were differentially expressed in head and neck squamous cell carcinoma.Through regression analysis,12 Lnc RNAs most related to the patient prognosis were obtained,including CASC9 whose expression was significantly up-regulated.2.CASC9 was overexpressed in head and neck squamous cell carcinoma tissues and predicts poor survival.3.There were 274 nasopharyngeal carcinoma DELnc RNAs in both data sets,including CASC9,which is significantly up-regulated.The Second Part: Expression and Clinical Significance of CASC9 inNasopharyngeal CarcinomaMethod1.Collect clinical samples,detect CASC9 expression,and analyze the relationship between CASC9 and the overall survival.2.Detect the expression of Lnc RNA CASC9 in nasopharyngeal carcinoma cell lines HNE-1,HONE-1,CNE-2,5-8F by q RT-PCR.Results1.The expression of CASC9 in NPC is increased,and it is related to the poor prognosis.2.Lnc RNA CASC9 is highly expressed in four nasopharyngeal carcinoma cell lines.The Third Part: The Function of CASC9 in NasopharyngealCarcinomaMethod1.Detect the proliferation of cells after silencing and overexpression of CASC9 by MTT and clone formation.2.Flow cytometry to detect the cell cycle of nasopharyngeal carcinoma cells after silencing and overexpression of Lnc RNA CASC9.3.Detect the migration and invasion of cells after silencing and overexpression of CASC9 by Transwell and wound healing test.4.MTT was used to detect the drug sensitivity of nasopharyngeal carcinoma cells after silencing and overexpression of CASC9 to cisplatin and paclitaxel.5.Detect the expression of proliferation,invasion and migration related proteins in NPC cells after silencing and overexpression of CASC9 by Western Blot.6.Detect the effect of silencing Lnc RNA CASC9 on the growth of nasopharyngeal carcinoma by nude mouse tumor formation experiment,and the expression level of Ki67 protein in tumor tissues.Results1.Silencing CASC9 would inhibit the proliferation of NPC cells,overexpression of CASC9 promote the proliferation.2.Silencing CASC9 induce G0/G1 arrest of NPC cells,overexpression of CASC9 promote cell cycle.3.Silencing CASC9 can inhibit the migration and invasion of NPC cells,overexpression of CASC9 promote the migration and invasion.4.Silencing CASC9 can improve the drug sensitivity of NPC cells to cisplatin and paclitaxel.5.Silencing Lnc RNA CASC9 can inhibit the protein expression of Ki67,PCNA,Cyclin D1,Cyclin E1,MMP2,MMP9,MDR1,MRP1 in nasopharyngeal carcinoma cells,and overexpression of Lnc RNA CASC9 can increase their expression levels.6.Silencing CASC9 can inhibit tumor growth in nude mice,and decrease the expression of Ki67 protein in tumor tissues.The Fourth Part: Mechanism of CASC9 Regulation of theOccurrence and Development of Nasopharyngeal CarcinomaMethod1.Detect the localization of Lnc RNA CASC9 in nasopharyngeal carcinoma cells by RNA FISH.2.Predict the target genes of CASC9 and mi R-497-5p.3.Verify the target genes of Lnc RNA CASC9 and mi R-497-5p through dual luciferase experiments.4.q RT-PCR detects the expression of mi R-497-5p and Wnt3 a.5.Detect the proliferation,cycle,migration,invasion of NPC cells and the expression of related proteins after recover mi R-497-5p.6.Western Blot detects the activity of Wnt3a/β-catenin pathway after over-expression of mi R-497-5p.Results1.RNA FISH shows that Lnc RNA CASC9 is distributed in both nucleus and cytoplasm of nasopharyngeal carcinoma cells.2.Mi R-497-5p is the target gene of CASC9,and Wnt3 a is the target gene of mi R-497-5p.3.The double luciferase reporter gene experiment verified that mi R-497-5p is the target gene of Lnc RNA CASC9,and Wnt3 a is the target gene of mi R-497-5p.4.The q RT-PCR showed that Lnc RNA CASC9 negatively regulates the expression of mi R-497-5p,and mi R-497-5p negatively regulates the expression of Wnt3 a.5.The recovery experiment showed that knocking down the expression of mi R-497-5p can reverse the effect of silencing Lnc RNA CASC9 on the proliferation,cycle,migration,invasion and related protein expression of nasopharyngeal carcinoma cells.6.Western Blot indicated that the overexpression of mi R-497-5p affects the expression of Wnt3a/β-catenin pathway related proteins.Conclusion1.CASC9 was overexpressed in nasopharyngeal carcinoma tissues and predicts poor survival;2.Silencing CASC9 may inhibit the proliferation,invasion and migration of nasopharyngeal carcinoma cells and the ability of tumor formation in nude mice,and may change the sensitivity to chemotherapy drugs;3.CASC9 might regulate the malignant biological behavior of nasopharyngeal carcinoma cells by targeting mi R-497-5p/Wnt3a/β-catenin signaling pathway.