The Effects And Mechanisms Of Ovarian Cancer Cells Stemness Regulated By Small Nucleolar RNA-SNORD89

Objective: Ovarian cancer is one of the common gynecologic oncology,since patients with early stage ovarian cancer do not have symptoms of discomfort and more than 70% of patients have been found in advanced stage(stage III or IV).Therefore,although the incidence rate of ovarian cancer is not very high,it is still the most lethal gynecologic oncology.According to WHO statistics,295414 cases of ovarian cancer will be diagnosed annually,and 184799 patients will die from this disease.The incidence rate and mortality rate of patients with ovarian cancer have a significant share in female tumors all over the world.In addition,ovarian cancer,especially advanced ovarian cancer,is highly resistant to radiotherapy and chemotherapy.Therefore,most of ovarian cancer patients have poor prognosis.The incidence rate of ovarian cancer is 3.4% of female tumors,and the mortality rate is up to 4.4%.With the development of research,the cancer stem cells(CSCs)theory points out a new direction and provides a new method for the treatment of ovarian cancer.The concept of CSC is like the origin of cancer cells,therapeutic resistance and immune system.Cancer stem cells(CSCs)are a kind of tumor cells with stronger self-renewal ability,unlimited proliferation ability,invasion and migration ability compared with ordinary tumor cells,which play an important role in tumor growth and recurrence.It is closely related to tumor recurrence,tumor cell dormancy and metastasis after successful treatment.CSCs have been reported in various tumors,such as leukemia,breast cancer,rectal cancer,brain cancer,etc.In recent studies,ovarian cancer is defined as "stem cell disease".CSCs can interact with tumor microenvironment,EMT and T cells to enhance tumor growth and drug resistance of tumor treatment.Small nucleolar RNA(SNORNA)is a kind of small non coding RNA,which is widely distributed in the nucleolus of eukaryotic cells.It has a stable metabolism and a length of 60-300 nucleotide sequences,and mainly divided into box C / D snoRNA and box H / ACA snoRNA.SNORNA could combine with specific proteins in nucleolus to form small nucleolar ribonucleoproteins partical(SNORNP)to perform post transcriptional processing and modification of ribosomal RNA(rRNA)and other RNA,such as pseudouridine modification and 2'-methylation modification.SNORNA has been considered as the noise in RNA transcription process,however,with the development of research,it has been found that the disorder of SNORNA is closely related to the occurrence,development and prognosis of a variety of tumors.The subgroups of snoRNA,such as SNORNA42,SNORNA78,SNORNA 113-1,SNORNA ACA11,SNORNA93,and SNORNA U2-19,have abnormal expression and regulation in various of tumors,and have effect on the occurrence and development of various tumors in different ways.For example,in lung cancer,the expression of SNORA42 is related to the expression of core transcription factors of lung cancer stem cells;SNORD14D and SNORD35 A play a key role in maintaining the activity of leukemia stem cells.Therefore,SNORNA has become a hot topic in tumor research.At present,there is no research and report of SNORNA in ovarian cancer.Notch1 encodes a class of highly conserved membrane protein receptors.Notch1 signaling pathway can affect many processes of cell biological behavior,especially cell functions closely related to tumor development,such as differentiation,apoptosis and proliferation of pluripotent stem cells.It has been reported that the cancer promoting effect of Notch1 is related to c-Myc,which can be used as a direct target of Notch1 in various of tumors.Many genes affect the progression of tumor by regulating Notch1 or c-Myc.However,there is a lack of research on the correlation between SNORNA and Notch1.Therefore,we screened out the SNORNAs that affects the prognosis of ovarian cancer patients by analyzing the data of 379 ovarian cancer patients in TCGA database.And,we detected the influence of the prognostic SNORNA on the stemness and biological behavior of ovarian cancer cells by in vitro experiment.Finally,we found that SNORD89 can affect the occurrence and development of ovarian cancer by targeting Notch1/c-Myc pathway.Methods: The clinical data and RNA-Seq data of 379 patients with ovarian cancer in TCGA database were analyzed.The high expression and prognosis related SNORNAs were screened out by using Log-rank(Mantel-Cox).The influence of SNORNA on the prognosis of different stages of ovarian cancer patients was compared.The relationship between the expression of SNORNA and the clinicopathological parameters of patients with ovarian cancer was analyzed by chi-square test and unpaired t-test,and the prognostic clinicopathological parameters of patients with ovarian cancer were selected by univariate survival analysis and multivariate survival analysis.Ovarian cancer stem cells(ovcar-3s,OS)were induced by ovarian cancer adherent cells(OVCAR-3,OV)through serum-free non adherent suspension culture.The expression of stemness markers in ovarian cancer stem cells was detected by agarose gel electrophoresis and flow cytometry.The high expression of SNORNAs in ovarian cancer stem cells were screened through the gene chips of normal epithelial cells(hosepic),ovarian cancer cells(OV)and ovarian cancer stem cells(OS).The expression of SNORNA was verified by qRT-PCR and agarose gel electrophoresis.The over expression of OV cells,CA cells and silencing OS cells were constructed by transfecting the over expression plasmids and silencing plasmids of SNORNA in OV,CA and OS cell lines respectively,and the efficiency of over expression and silencing was determined by qRT-PCR.The expression of stemness markers in OV cells and OS cells after the interference of SNORNA was detected by q RT-PCR,agarose gel electrophoresis and flow cytometry.Flow cytometry was used to detect the changes of cell cycle in ovarian cancer cells after interfering with the expression of SNORNA.The effects of overexpression and silencing of SNORNA on the proliferation and malignancy of ovarian cancer cells were detected by CCK-8 cell proliferation experiment,plate clone formation experiment and soft agar clone formation experiment.The effect of SNORNA silencing on the self-renewal ability of ovarian cancer stem cells was detected by colony formation assays.Scratch test and migration test were used to detect the effect of interfering with SNORNA on the invasion and migration of ovarian cancer cells and ovarian cancer stem cells.qRT-PCR,agarose gel electrophoresis and western blot were used to detect the expression of Notch1 and c-Myc genes and protein expression in ovarian cancer cells,ovarian cancer stem cells.Results:1.The high expression of SNORD89 and SNORD116-4 is associated with poor prognosis of ovarian cancer patients.Survival analysis according to stage showed that the high expression of SNORD89 in stage ? patients and stage ? patients related to poor prognosis,moreover,the effect of SNORD89 on the prognosis of ovarian cancer patients is greater than that of clinical stage of ovarian cancer.The high expression of SNORD116-4 suggested that the poor prognosis of stage ? patients,however,patients with high expression of SNORD116-4 in stage IV have better prognosis.Therefore,SNORD116-4 can't indicate the prognosis of ovarian cancer patients.The expression of SNORD89 is related to the age and therapeutic effect of ovarian cancer patients.SNORD89 expressed more higher in the elderly patients and poor therapeutic effect.Single factor and multi factor Cox survival analysis were used to analyze the prognostic factors of ovarian cancer patients.The expression of SNORD89,the age of patients and the size of tumor were the dependent risk factors of the overall survival time of ovarian cancer patients.The race of patients and the lymph node metastasis of tumor were the independent risk factors of the overall survival time of ovarian cancer patients.Age is a dependent risk factor for progression free survival time,and lymph node metastasis is an independent risk factor for progression free survival time.2.The results of gene chips,qRT-PCR and agarose gel electrophoresis experiments showed that the expression level of SNORD89 in ovarian cancer stem cells was higher than that in ovarian cancer cells and ovarian epithelial cells.The expression of CD133,CD44 and Nanog were high in ovarian cancer cells.The expression of stemness markers increased after overexpression of SNORD89 and decreased after silencing SNORD89.3.SNORD89 co-expressed with some genes related to cell cycle.The proportion of S phase ovarian cancer cells increased after overexpression of SNORD89,while that cells in G1 phase and G2 phase decreased;the proportion of ovarian cancer cells in G1 phase and S phase decreased after silencing SNORD89,and that in G2 phase increased.4.The proliferation and malignancy of ovarian cancer cells were enhanced after overexpression of SNORD89,and reduced the proliferation and self-renewal of ovarian cancer stem cells after silencing SNORD89.The invasion and migration ability of ovarian cancer cells were improved after overexpression of SNORD89,and reduced the migration ability of ovarian cancer cells after silencing SNORD89.5.The expression at gene and protein levels of Notch1 and its target gene c-Myc increased after overexpression of SNORD89,and decreased after silencing SNORD89.Conclusions:1.The expression of SNORD89 is closely related to the prognosis of patients with ovarian cancer and positively related to the age of patients.2.Ovarian cancer stem cells with self-renewal ability,invasion and migration ability,differentiation potential and high expression of stemness markers can be successfully enriched by serum-free non adhesion suspension culture of ovarian cancer cells.3.SNORD89 was highly expressed in ovarian cancer stem cells.4.SNORD89 promotes the stemness of ovarian cancer cells.5.SNORD89 can interfere with the cell cycle of ovarian cancer cells,and promote the proliferation,self-renewal,invasion and migration of ovarian cancer cells.6.SNORD89 promotes the stemness phenotype of ovarian cancer cells by targeting the Notch1/c-Myc pathway.