| Objective:The microarray data of high throughput sequencing in Gene Expression Omnibus(GEO)database were analyzed by bioinformatics method.From the differentially expressed gene(DEGs)between cervical cancer tissue samples with and without lymph node metastasis(NO),the secretory carrier membrane protein 1(SCAMP1)involved in membrane vesicle transport was screened and further predicted by online tools.To obtain miRNA-miR-194-5p that may regulate SCAMP1.Based on the above analysis results,we further detected the expression levels of miR-194-5p and SCAMPI in cervical cancer cell lines,,cervical cancer tissue samples from cervical cancer patients with N+and N0.We then planed to explore the effects of miR-194-5p and SCAMP1 on the migration and invasion of cervical cancer cells.Finally,we planed to investigate the direct evidence of miR-194-5p targeting SCAMP1 gene in cervical cancer cell lines.Methods:1.The microarray data of high-throughput sequencing in Gene Expression Omnibus(GEO)database were analyzed by bioinformatics method,and the deferentially expressed genes(DEGs)and minimal RNA(DEMs)were obtained between cervical cancer tissues with(N+)and without(N0)lymph node metastasis.The genes related to membrane vesicle transport,SCAMP1,were screened from DEGs.The miRNA(miR-194-5p)which may regulate SCAMP1 was obtained by online tools.2.Collection of postoperative tissue samples of cervical cancer:66 cases of postoperative tissue samples of patients with cervical cancer underwent primary surgery in Yunnan Cancer Hospital from February 2019 to August 2019 were collected,including 13 cases of N+tissue samples and 53 cases of NO tissue samples.6 tissue samples were collected from each patient.3.The differential expression of miR-194-5p and SCAMP1 between N+and NO full transcripts was detected by high-throughput sequencing in 4 cases of N+and 4 cases of N0,respectively,and the differential expression results of miR-194-5p and N+obtained from NO database were verified again.4.RT-qPCR method was used to detect the expression of SCAMP1 mRNA and miR-194-5p in various human cervical cancer cell lines(Hela,Cmur33A,Ca Ski,SiHa)and normal cervical epithelial cell line(HcerEpic),cervical cancer tissues and paracancerous tissues(12 cases of cervical cancer and corresponding para-cancerous tissues),as well as N+and NO tissues(8 cases each).5.6 cases of NO and 6 cases of NO cervical cancer were selected,and the protein was extracted,and the protein expression of SCAMP1 protein was detected by Western blotting(WB).6.6 cases of NO and 6 cases of N0 cervical cancer were selected,and the expression of SCAMP1 in NO and N0 cancer tissues was detected by immunohistochemical staining(IHC)and compared.Results:1.The analysis of GSE7410 and GSE102969 microarray datasets in GEO database showed that compared with NO tissue,the expression of SCAMPI in NO tissue was up-regulated and logFC=0.26,(p<0.05),while the expression of miR-194-5p was down-regulated in NO tissue and logFC=-2.24,(p=0.047).31 miRNA,targeting SCAMP1 were predicted by using TargetScan and starBase database.Among them,miR-194-5p is highly conservative and most likely to directly regulate the upstream miRNA;of SCAMP1.2.A total of 66 postoperative tissue samples of cervical cancer were collected,including 13 NO tissue samples and 53 NO tissue samples,and 6 tissue samples were collected from each patient.3.The sequencing results showed that there were 59 deferentially expressed genes between NO and NO,including 24 up-regulated and 35 down-regulated(|log2FC|>1,FDR<0.05),62 up-regulated and 41 down-regulated in miRNA,(|log2FC|>2,p<0.05).The results of sequencing showed that there was no difference in the expression of SCAMP1 between NO and N+(log2(FC)=-0.41),and there was no significant difference in the expression of miR-194-5p(log2(FC)=1.4).4.At the level of cervical cancer cell lines,the expression of miR-194-5p in cervical cancer cells was higher than that in normal cervical epithelial cells,the expression of miR-194-5p in C-33A,Ca-Ski and SiHa cells was higher than that in HcerEpic cells,and the expression of SCAMP1 was also up-regulated in C-33A and SiHa cells.At the tissue level,the results of qRT-PCR detection showed that there was no difference in the expression of miR-194-5p and SCAMP1 in cervical cancer tissues compared with adjacent tissues,but the expression of SCAMP1 in N+tissues was significantly up-regulated compared with NO tissues(p=0.015),while there was no difference in miR-194-5p between NO and NO tissues5.The results of Western blotting showed that there was no significant difference in the expression of SCAMP1 between NO and NO(p=0.11).6.The expression of SCAMP1 in 6 cases of NO and 6 cases of NO+cervical cancer was detected by immunohistochemistry.It was found that SCAMP1 was expressed in both NO and NO+tissues,but the the difference of staining intensity of SCAMP1 in both N0 and NO+tissues was not significant.Conclusions:1.GSE7410 analysis of the data set showed that compared with NO tissue,the expression of SCAMP1 in NO tissue was significantly up-regulated and miR-194-5p was down-regulated in NO tissue,but the sequencing results showed that there was no difference in the expression of miR-194-5p and SCAMP1 between NO and NO tissue.2.Compared with normal cervical epithelial HcerEpic cells,miR-194-5p was highly expressed in C-33A,Ca-Ski and SiHa cells,while SCAMP1 expression was significantly up-regulated in Cervical carcinoma Cmur33A and SiHa cells.3.At the RNA level,there was no difference in the expression of miR-194-5p and SCAMP1 in cervical cancer tissues and paracancerous tissues,but compared with NO tissues,the expression of SCAMP1 was significantly up-regulated in N+tissues,while there was no difference in miR-194-5p between N+and NO tissues.At the protein level,there was no significant difference in the expression of SCAMP1 between N+ and N0 tissues.4.In short,the expression of miR-194-5p and SCAMPI in cervical cancer cells and tissues is inconsistent,and they may have no effect on lymph node metastasis of cervical cancer. |
PDF Full Text Request