| Objectives:To analyze the differentially expressed m RNA and lnc RNA among prostatic carcinoma(PCa),and their paired adjacent noncancerous tissues by microarray,with the aim to find out the role of LAMA4 gene and relevant lnc RNA for refreshing molecular mechanism and providing new proposal in the preservation,clinical diagnosis,treatment and pathogenesis of prostate cancer.Methods:First of all,we collected 20 clinical tissues from prostatectomy between August 2013 and August 2014 in our hospital and established the extraction methods of high-quality total RNA from clinical tissues by improve obtaining,transportation and grinding of samples.Then,the purity of PCa,PIA and their paired adjacent noncancerous clusters in the same prostate were isolated by cryosections combined with manual microdissection technology.In the end,we performed bioinformatic analysis of them by combining lnc RNA expression profiling and m RNA expression profiling.The Pathway analysis and targets prediction using reversing Cis were performed,meanwhile,we constructed a genomic network of lnc RNA and its target genes in some inflammation and/or cancer signaling pathways,related to the differentially expression profiling.For identifying the differential expression of LAMA4 and its relevant lnc RNA,we have collected 6 clinical samples from prostatectomy between November 2014 and October 2015 in our hospital,with the methods of PCR-examination,culturing cell lines of DU145 and 22RV1,transfecting with specific si RNA,wounding assay,transwell assay and cell adhesion assay by MTT method,in order to investigate the role of lnc RNA in the metastasis and adhesion of tumor cells.Results:1.Of the 2610 m RNA dysregulated detected in the human PCa tissues when compared to adjacent noncancerous tissues,689 genes were up-regulated and 1921 genes were down-regulated.At the same time,2176 lnc RNA were differentially expressed,with 688 up-regulation and 1488 down-regulation.Compared with adjacent noncancerous tissues,183 of 592 m RNA profiles were up-regulated and 409 of 592 were down-regulated in human PIA tissues,meanwhile,575 lnc RNA were differentially expressed,with 160 up-regulation and 415 down-regulation.Of 1007 m RNA dysregulated between the PCa tissues and the PIA tissues,115 genes were up-regulated and 892 genes were under-regulated.773 lnc RNA were differentially expressed as well,105 lnc RNA of them were up-regulated and 668 lnc RNA of them were under-regulated.The 113 overlapping m RNA showed significant linear difference among them,8 genes were commonly up-regulated and 105 genes were commonly down-regulated.Also,The 106 overlapping lnc RNA showed significant linear difference among them,11 genes were commonly up-regulated and 95 genes were commonly down-regulated.2.LAMA4 and its relevant lnc RNA have significantly different expression in the microarray,and the result of tissue PCR was in accordance of that of microarray.3.After the transfection,the result of PCR demonstrated the specific si RNA has down-regulated lnc RNA and m RNA of LAMA44.Cell functional assay showed the transfected cell has low capability of metastasis and adhesion.Conclusions:1.It is an economical and valid method to qualify and isolate targeted cell sub-lines by H&E staining of cryosections combined with the “elimination” manual microdissection,which were used for extracting high-quality RNA,by Trizol,consistent with the standards of high-throughput microarray technology.2.PCR-examination of tissue were in accordance with microarray chips,LAMA4 and its relevant lnc RNA were increased in the cancer tissue compared with adjacent noncancerous tissue(P<0.05)..3.RT-PCR and cell adhesion assay have demonstrated that the transfected cells have lower level of LAMA4 and its relevant lnc RNA,and the capability of metastasis,migration and adhesion have been prohibited. |
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