Mechanism Of CASC9 Gene Regulating The Progression Of Nasopharyngeal Carcinoma By Targeting MiR-145/SMAD4 Signal

BackgroundNasopharyngeal carcinoma is a common malignant tumor in otolaryngology,which occurs in nasopharyngeal epithelial cells.The preferred treatment method commonly used in clinical practice is radiotherapy,but there are many adverse reactions,which cause great trouble to patients.Therefore,it is of great clinical significance to deeply study the pathogenesis of nasopharyngeal carcinoma and find new therapeutic targets and drugs.Non-coding RNA(nc RNA)covers more than 98% of the human genome.Among them,miRNA is a non-coding micro RNA with a length of 19-23 nucleotides.Mi RNA plays an important role in the occurrence and development of cancer.Long non-coding RNAs(lnc RNAs)participate in various pathophysiological processes in humans.Long-chain non-coding RNAs have been studied in cancer in recent years increasingly,and their functions have been almost as positive as miRNAs.Long-chain non-coding RNA CASC9 is a recently discovered lnc RNA that plays a regulatory role in some cancers.Its research in nasopharyngeal carcinoma is rare,and its molecular mechanism has not been fully understood at home and abroad.This subject includes the following three parts: The first part: the expression and clinical significance of CASC9 gene in nasopharyngeal carcinoma;the second part: the mechanism of CASC9 gene regulation of nasopharyngeal carcinoma cell process;the third part: Silencing CASC9 inhibits tumor growth of nasopharyngeal carcinoma in vivo.Part One The expression of CASC9 gene in nasopharyngeal carcinoma and its clinical significance Method:1.The expression level of CASC9 gene in nasopharyngeal carcinoma and nasopharyngeal carcinoma cell lines HNE1,SUNE1,CNE1 and HONE1 was detected by real-time PCR.2.Retrospectively investigated the relationship between the expression of CASC9 gene and the overall survival time of patients with nasopharyngeal carcinoma.Result:1.The expression level of CASC9 gene in nasopharyngeal carcinoma tissues was significantly increased,and was closely related to the staging and metastasis of patients.2.The expression level of CASC9 gene in nasopharyngeal carcinoma cell lines HNE1,SUNE1,CNE1 and HONE1 was significantly increased.3.The high expression of CASC9 gene in nasopharyngeal carcinoma tissues was inversely related to the overall survival time of patients.Part Two The mechanism of CASC9 gene regulation of nasopharyngeal carcinoma cell process Method:1.The proliferation of nasopharyngeal carcinoma cells after silencing CASC9 and returning to miR-145 was detected by MTT assay.2.Transwell method was used to detect the migration and invasion of nasopharyngeal carcinoma cells after silencing CASC9 and returning to miR-145.3.Cyclic blockade and apoptosis of nasopharyngeal carcinoma cells after silencing CASC9 and returning to miR-145 were detected by flow cytometry.4.The protein expression of MMP2,MMP9,Bcl-2 and Bax in nasopharyngeal carcinoma cells after silencing CASC9 and returning miR-145 was detected by Western blot.5.Bioinformatics analysis speculated the target genes of CASC9 and miR-145.6.Recombinant plasmid of wild and mutant CASC9 and recombinant plasmid of SMAD4 were constructed,and the target gene of CASC9 and the target gene of miR-145 were verified by dual luciferase reporter assay and RNA immunoprecipitation assay.7.The expression of miR-145 was detected by real-time PCR after overexpression and knockdown of CASC9,and the expression of SMAD4 was detected by real-time PCR after overexpression and knockdown of miR-145.Result:1.Silencing CASC9 can inhibit the proliferation of nasopharyngeal carcinoma cells,and it can promote the proliferation of nasopharyngeal carcinoma cells after returning to miR-145.2.Silencing CASC9 can inhibit the migration and invasion of nasopharyngeal carcinoma cells,and it can promote the migration and invasion of nasopharyngeal carcinoma cells after returning to miR-145.3.Silencing CASC9 can block the G1 phase of nasopharyngeal carcinoma cells and promote the apoptosis of nasopharyngeal carcinoma cells.After reverting to miR-145,it can promote the nasopharyngeal carcinoma cell cycle and inhibit the apoptosis of nasopharyngeal carcinoma cells.4.Silencing CASC9 can inhibit the expression of migration and invasion marker proteins MMP2 and MMP9,inhibit the expression of Bcl-2,and promote the expression of Bax.5.Bioinformatics prediction showed that miR-145 is the target gene of CASC9,and SMAD4 is the target gene of miR-145.6.Double fluorescent reporter gene and RNA immunoprecipitation experiments confirmed that miR-145 is the target gene of CASC9,and SMAD4 is the target gene of miR-145.7.CASC9 negatively regulates the expression of miR-145,and miR-145 negatively regulates the expression of SMAD4. Part Three Silencing CASC9 inhibits tumor growth of nasopharyngeal carcinoma in vivo Method:1.The nasopharyngeal carcinoma cell CNE1 silencing the CASC9 gene was constructed by lentivirus.2.The effect of silencing CASC9 gene on tumor growth was detected by nude mouse tumor formation assay.3.Real-time PCR was used to detect the effect of silencing CASC9 gene on the expression of miR-145 and SMAD4 in tumors.4.The expression of MMP2,MMP9,Bcl-2 and Bax expression in tumors was detected by Western blot.Result:1.Successfully constructed CNE1 cells with lentivirus-mediated CASC9 gene silencing.2.Silencing CASC9 gene can inhibit tumor growth in nude mice.3.Silencing CASC9 gene can inhibit the expression of migration and invasion marker proteins MMP2 and MMP9,inhibit the expression of Bcl-2 and promote the expression of Bax.Conclusions1.CASC9 gene is highly expressed in nasopharyngeal carcinoma and is associated with the staging and prognosis of nasopharyngeal carcinoma.2.CASC9 gene can regulate the proliferation,migration,invasion,cell cycle and apoptosis of nasopharyngeal carcinoma cells through miR-145/SMAD4 pathway.3.CASC9 gene can regulate tumor growth by regulating the expression of miR-145 and SMAD4 in nude mice.