| IntroductionThere are more than 400000 cases of renal cell carcinoma(RCC)every year in the world,and about 70%of them are clear renal cell carcinoma(ccRCC).They could be successfully treated by surgery or ablation in the early stage,but 30%of the patients would progress to advanced stage or were initially diagnosed with metastasis.Long non coding RNA(lncRNA)is a member of the family of non-coding RNAs,with a minimum length of 200 nucleotides and does not encode proteins.With the popularization and application of sequencing technology,a large number of lncRNAs have been found in different cancers with abnormal expression.Many studies have confirmed that abnormal levels of lncRNAs affects the biological function of tumor.In renal cell carcinoma,a variety of lncRNAs have been found to participate in the formation and development of tumor.Although there are few reports on the function of LINC01094 in renal cell carcinoma,this study will further clarify the biological function of LINC01094,and find the miRNAs and mRNAs that may be targeted,explore the possibility of LINC01094 as a potential biomarker and therapeutic target in renal cell carcinoma,with these can provide ideas for later clinical application.Object1.Explore the differentially expressed lncRNAs in renal cell carcinoma2.Clarify the clinical significance of LINC01094 in renal cell carcinoma3.Explore the biological function of LINC01094 in renal cell carcinoma4.Explore the molecular mechanism of LNC01094 in the lncRNA-miRNA-mRNA network systemMethodsPart Ⅰ LINC01094 is up-regulated in RCC and promotes the growth of RCC cells1.Analyze gene expression datasets by bioinformatics software to found differentially expressed genes.2.RNA were extracted from 56 cases of renal cell carcinoma,then detect the expression level of LINC01094 in the cancer and adjacent normal tissues by RT-qPCR.3.Analysis the clinicopathological data of patients with renal cell carcinoma,divide the patients into low expression group and high expression group according to the level of LINC01094,explore the relationship between LINC01094 and the prognosis of patients with using univariate and multivariate analysis.4.Screen renal cell carcinoma cell lines for further study.Using sh-LINC01094 to knock down or oe-LINC01094 to over express the level of LINC01094,then test the effects of LINC01094 on cell proliferation,migration and invasion,using experiments of CCK-8,cell scratch test and Transwell cell invasion test,also detect the cell apoptosis ability by flow cytometry.Part Ⅱ Interaction between LINC01094 and the target of miR-1841.Predict the targeted binding miRNAs of LINC01094 by using the bioinformatics databases.2.To verify the interaction between LINC01094 and miR-184,Luciferase reporter gene assay,RNA binding protein immunoprecipitation assay and RNA pull-down assay were used.3.Use RT-qPCR to detect the expression levels of miR-184 in renal cell carcinoma and normal tissues adjacent to cancer,also in renal cell carcinoma cell lines.The function of miR-184 were tested in renal cell carcinoma cell by CCK-8 cell proliferation test,cell scratch test,Transwell cell invasion test and flow cytometry,the cells was transfected with miR-184 mimics/inhibitor.4.Transfected with sh-LINC01094/oe-LINC01094 and miR-184 mimic/inhibitor in renal cell carcinoma cell lines,the changes in proliferation,migration,invasion and apoptosis were detected by every experimental techniques.Part Ⅲ LINC01094 up-regulates the expression of SLC2A3 by targeting miR-1841.Bioinformatic tools were used to explorer the target genes of miR-184.2.RT-qPCR and Western blot were used to detect the expression of SLC2A3 in RCC and adjacent normal tissues.3.The interaction between SLC2A3 and miR-184 was detected by luciferase reporter assay;4.Sh-SLC2A3 knockdown and oe-SLC2A3 overexpression combined with miR-184 mimic were used to treat the cells,then the changes in proliferation,migration,invasion and apoptosis were detected by experimental techniques.5.In the xenograft tumor model,renal cell carcinoma cells treated with oe-LINC01094 overexpression and sh-LINC01094 knockdown,then the RNA levels of miR-184 and SLC2A3 in tumor tissue was detected by RT-qPCR,and the protein expression of SLC2A3 was detected by Western blot.ResultsPart Ⅰ1.Bioinformatics analysis indicated that 3776 genes were differentially expressed,including 886 lncRNAs in GSE53757.According to the differentially expressed genes,the corresponding heat maps were drawn.Among the up-regulated lncRNAs,we found that the expression of LINC01094 was significantly higher in cancer tissues than that in adjacent normal renal tissues(ID:229635_at,logFC:=2.549,adj.P.Val=2.26E-30).2.RT-qPCR was used to detect the expression of LINC01094 in 56 cases of renal cell carcinoma and adjacent normal tissues.The average relative expression level of LINC01094 in renal cell carcinoma was(2.18±0.30),which was significantly higher than that in adjacent normal tissues(1.00±0.16),also the difference was statistically significant(p<0.05).3.The expression of LINC01094 was not related to gender,age and tumor size(p>0.05),but related to TNM stage,Fuhrman grade,the status of vascular invasion and lymph node metastasis(p<0.05).Survival analysis showed that the metastasis free survival time of patients with low expression of LINC01094 was significantly longer than that with high expression(p<0.05).4.LINC01094 was highly expressed in all renal cell carcinoma cell lines,with the highest expression in 786-O cells.Fluorescence in situ hybridization showed that LINC01094 was localized in the cytoplasm of 786-O cells.When sh-LINC01094 was used to knock down the expression of LINC01094 in 786-O cells,the proliferation of 786-O cells was significantly inhibited,the ability of migration and invasion was significantly decreased,and the number of apoptosis was increased.These results indicate that LINC01094 plays a role in promoting growth of renal cell carcinoma.Part Ⅱ1.Bioinformatics predicted that one targeted miRNA of LINC01094 was miR-184.Dual luciferase reporter gene experiment showed that compared with the control group,the relative luciferase activity in LINC01094-WT reporter plasmid treated group and miR-184 mimic co-transfection group was decreased,while the relative luciferase activity of LINC01094-MUT group did not change,indicating that there were complementary binding sites between miR-184 and LINC01094 sequences.2.RIP experiment in 786-O cell line indicated that the relative expression level of LINC01094 in the precipitation complex in Ago2 group was higher than that in IgG group,and the relative expression level of LINC01094 in miR-184 inhibitor group was lower than that in Ago2 group.Using biotin labeled RNA probe biotin-miR-184 for RNA pull-down experiment,compared with blank control group and biotin-miR-184 mutant group,the expression enrichment of LING01094 in biotin-miR-184 wild type probe group was significantly increased.These results indicated that LINC01094 could sponge miR-184.3.The expression of miR-184 was lower in RCC than in adjacent tissues.RT-qPCR showed that the expression of miR-184 was lower in every renal cell carcinoma cell line than in HK-2 cells,and the lowest expression of miR-184 was in 786-O cells.After transfected with miR-184 mimics,the proliferation activity of cells was significantly inhibited,the migration and invasion ability were significantly decreased,but the number of apoptosis cells was increased.These results indicate that miR-184 plays an anti-tumor role in renal cell carcinoma.4.After sh-LINC01094 transfected to knockdown,the expression of miR-184 in 786O cell was significantly increased,while transfected with oe-LINC01094 overexpression,the expression of miR-184 was lower than that in the control group.Compared with the control group,if treated by miR-184 mimics,the expression of LINC01094 decreased,when miR-184 inhibitor added,the expression of LINC01094 could increased significantly.In cotransfection group of sh-LINC01094 and miR-184 NC inhibitor,the expression of LINC01094 decreased,the expression of miR-184 was significantly increased.In the reversal experiment,the expression of LINC01094 was down regulated by sh-LINC01094,and the expression of miR-184 was significantly increased.After addition with miR-184 inhibitor,the expression of LINC01094 was reversed.These results indicate that LINC01094 negatively regulates the expression of miR-184.5.CCK-8 proliferation assay showed that sh-LINC01094 transfected cells had the weakest proliferation ability.After transfection of miR-184 inhibitor,the proliferation ability recovered.Cell scratch test and Transwell invasion test also showed that the migration and invasion ability of LINC01094 knockdown cells was significantly decreased,while the migration and invasion ability of cells treated with miR-184 inhibitor was restored.Flow cytometry showed that down-regulation of LINC01094 significantly promoted cell apoptosis,while miR-184 inhibitor could partially reverse the numbers of apoptosis induced by sh-LINC01094.These results indicate that LINC01094 interacts with miR-184 and affects the biological behavior of renal cell carcinoma cells.Part Ⅲ1.The downstream target genes regulated by miR-184 were predicted,SLC2A3 was screened for subsequent study.Bioinformatics predicted that there were binding sites between miR-184 and SLC2A3.2.The results of dual luciferase reporter gene experiment showed that compared with the control group,the relative luciferase activity of the wild-type reporter gene plasmid(SLC2A3-WT)and miR-184 mimic co-transfection group was lower,while the relative luciferase activity of the mutant plasmid(SLC2A3-MUT)and miR-184 mimic cotransfection group was basically unchanged,indicating that miR-184 can specifically bind to SLC2A3 gene.3.RT-qPCR and Western blot experiment were used to detect the expression of SLC2A3 in clinical samples.The results showed that the expression of SLC2A3 in cancer tissue was higher than that in adjacent normal tissue.The correlation analysis between the expression of SLC2A3 and miR-184 showed that R=-0.845,p<0.001,indicating the negative correlation between the expression of miR-184 and SLC2 A3 in renal cell carcinoma tissues.Immunohistochemical staining showed that SLC2A3 was mainly expressed in cytoplasm and cell membrane.4.After sh-SLC2A3 transfected to knockdown and oe-SLC2A3 transfected to overexpress,the expression levels of LINC01094,miR-184 and SLC2A3 mRNA and the protein expression of SLC2A3 were detected by RT-qPCR and Western blot.The results showed that the expression levels of SLC2A3 mRNA and protein were respectively significantly decreased and increased,but the expression levels of LINC01094 and miR-184 were not significantly changed.CCK-8 test,cell migration and invasion test showed that overexpression of SLC2A3 enhanced cell proliferation,invasion and migration,the apoptosis test showed that the number of apoptosis decreased vice versa.The above results showed that the expression of SLC2A3 did not affect the expression of LINC01094 and miR-184.Meanwhile,overexpression of SLC2A3 could significantly promote the proliferation,invasion and migration of renal cell carcinoma cells,and inhibit the apoptosis of renal cell carcinoma cells.5.After transfection of miR-184 mimic and oe-SLC2A3-NC,RT-qPCR showed the expression of miR-184 was significantly increased,while the expression of SLC2A3 was decreased by Western blot.CCK-8 test showed that the cell proliferation was decreased,scratch test and Transwell test showed that the cell migration and invasion ability were decreased,but flow cytometry showed that the number of apoptotic cells was increased.On the contrary,in miR-184 mimic with oe-SLC2A3 cotransfected group,cell proliferation,migration and invasion were restored,and the number of apoptotic cells was reduced.These results indicate that miR-184 plays a negative regulatory role in SLC2A3.6.Sh-LINC01094 was used to knock down the expression of LINC01094 in 786-0 cells.The stably LINC01094-low expression cell lines were selected and inoculated into nude mice.The results showed that the tumor growth rate of LINC01094-low expression group was slower than that of control group.On the contrary,oe-LINC01094 transfected 786-O cell grew faster than the control group.On the 28th day,the tumor volume of oeLINC01094 overexpression group and sh-LINC01094 knockdown group were 1581.8±108.2mm3 and 901.9±100.1mm3,respectively,and the weight were 1.52±0.18g and 0.36±0.05g,respectively(p<0.05).The further study on nude mice tumor showed that compared with the control group,the LINC01094 overexpression group has the increased expression of LINC01094 and SLC2A3,while with the decreased expression of miR-184.ConclusionLINC01094 is highly expressed in renal cell carcinoma tissues and cell lines.The expression level of LINC01094 is related to the Metastasis-free survival time of renal cell carcinoma patients.Knockdown of LINC01094 significantly inhibits the proliferation,migration and invasion of renal cell carcinoma cells,but promotes the apoptosis of tumor cells.LINC01094 targets miR-184 and interacts with miR-184 negatively.SLC2A3 is the target gene of miR-184,and knockdown of LINC01094 inhibits the expression of SLC2A3.The interaction among LINC01094 target miR-184 and SLC2A3 was also demonstrated in nude mice in vivo.In general,our study reveals that LINC01094 has potential as a prognostic marker of renal cell carcinoma.It also expounds that LINC01094 plays a role in regulating the growth and invasion of renal cell carcinoma cells through miR-184/SLC2A3,which provides new evidence and theoretical support for LINC01094 as a therapeutic target in the future. |
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