The Effects Of Long Non-coding RNA MIR4435-1HG In Renal Cell Carcinoma And Its Molecular Mechanism

Part ?:The expression of lncRNA MIR4435-1HG in renal cell carcinoma and its clinical significanceObjective:To detect the abnormally expressed long non-coding RNA(lncRNA)in renal cell carcinoma,and to explore the relationship between its expression level and clinical pathological parameters of renal cell carcinoma cases as well as and its influence on patients' prognosis.Methods:Three pairs of renal cancer specimens and adjacent tissues were selected.The expression of lncRNA in renal cancer and adjacent tissues was detected by Human LncRNA One Array Plus(Beijing Liuhe Huada Gene Technology Co.,Ltd.).Surgical resection of renal clear cell carcinoma and adjacent specimens were collected with all cases confirmed by pathology as clear cell carcinoma.The abnormally expressed lncRNA found by the microarray was analyzed by Gene Ontology illustration.The expression level of lncRNA was verified in cell lines and in tissues.Patients with renal cancer were followed up for prognosis.Cell lines and tissues were separately subjected to RNA extraction and PCR detection.The data was analyzed using SPSS Statistics 16.0.Results:The lncRNA microarray analysis showed a significant difference in lncRNA expression between kidney cancer and adjacent tissues.Among the overly expressed lncRNAs,the expression level of MIR4435-1HG in renal cell carcinoma was significantly higher in renal cell carcinoma than that in adjacent tissues.Go Ontology showed that MIR4435-1HG was located in mitochondria or intracellular membrane,and it was mainly involved in PI3K-Akt signaling pathway or regulation of microRNAs in tumors.The process which MIR4435-1HG involved was related to apoptosis.In this study,a total of 55 cases of renal clear cell carcinoma were enrolled.55 cases of renal cancer specimens were paired with adjacent tissues,and the expression level of MIR4435-1HG was detected by PCR after RNA extraction.The expression level of MIR4435-1HG in renal cancer tissues was higher than that in adjacent tissues.There was no significant difference with the expression level of MIR4435-1HG in renal cell carcinoma between males and females,p=0.113.There was no significant difference with the expression level of MIR4435-1HG in renal cell carcinoma cases at different ages,p=0796.The expression level of MIR4435-1HG In Fuhrman grade 3+4 renal cell carcinoma cases was higher than that in Fuhrman grade 1+2 renal cell carcinoma cases;p<0.001.The expression level of MIR4435-1HG in tumor size>50mm renal cancer tissues was higher than that in tumor size<50mm renal cancer tissues,p=0.0415.The expression level of MIR4435-1HG in T2-4N0-1M0-1 kidney cancer tissues was higher than that in T1N0M0 kidney cancer tissues,p<0.001.The recurrence-free survival of patients with MIR4435-1HG high expression was shorter than patients with MIR4435-1HG low expression,p=0.028.The overall survival time of patients with MIR4435-1HG high expression was not significantly different from patients with MIR4435-1HG low expression,p=0.127.The receiver operating characteristic curve(ROC)was plotted by SPSS,and the area under the ROC curve was 0.946(95%confidence interval:0.906-0.986).Conclusion:MIR4435-1HG is a long non-coding RNA abnormally expressed in renal cell carcinoma,and its expression level is significantly higher than that of adjacent tissues.In renal cell carcinoma,the expression level of MIR4435-1HG is not related to the age or the gender of the cases,but is related to Fuhrman grade,TNM stage and tumor size.High expression of MIR4435-1HG suggests poor prognosis in renal cell carcinoma patients.MIR4435-1HG can be used as a biomarker with promising diagnostic value.MIR4435-1HG plays a carcinogenic role in the development of renal clear cell carcinoma,and may serve as a new predictor for clinical prognosis of renal cancer.Part ?:Biological function of lncRNA MIR4435-1HG in renal carcinoma cellsObjective:To explore the effects of lncRNA MIR4435-1HG on the biological behavior of renal cell carcinoma cells,in this part we explored the biological function of MIR4435-1HG by in vitro experiments.Methods:Overexpression and knockdown of MIR4435-1HG was achieved by lentiviral vector constructed by Genecham Gene Co.,Ltd.Renal cancer cell lines and normal cell line were purchased from the Cell Bank of Academy of Sciences.HK-2,786-0,and OSRC-2 cells were cultured after resuscitation.Overexpression and knockdown efficiency were verified to select suitable lentiviral vectors for subsequent use.After treatment with lentiviral vector,RNA was extracted from the cells,and reverse transcription and PCR were performed.Cell proliferation assay was performed using the CCK-8 kit.Cell clones were detected and clone formation was calculated.Both cell apoptosis detection and cell cycle detection used Flow CytoMetry.Cell transwell assay was conducted to detect cell invasion and migrationResults:RT-PCR confirmed the effect of lentiviral vector on the expression of MIR4435-1HG.The expression level of MIR4435-1HG after over-expression virus transfection was significantly higher than that of NC group.Two kinds of knockdown lentvirus were used to infect cell lines,between which the expression level of MIR4435-1HG in the 70153-1 group was significantly lower than that in the control group Thus we used 70153-1 as a lentiviral vector to down-regulate the expression of MIR4435-1HG afterwards.The cell proliferation ability of MIR4435-1HG was detected by CCK-8 method:compared with the control group(NC),the cell proliferation rate of knockdown group(KD)decreased,p<0.05;compared with the control group(NC),the cell proliferation rate of over-expression group(OE)increased,p<0.05.The results of colony formation experiments showed that in the three cell lines,after lentivirus infection,the number of cell clone formation in the KD group was significantly reduced compared with the NC group,and the difference was statistically significant(p<0.05);compared with the NC group,the number of cell clone formation in the OE group was significantly increased,and the difference was statistically significant(p<0.05).The results of cell cycle assay showed that the proportion of cells in G0/G1 phase of KD group was significantly higher than that of NC group,and the proportion of cells in S phase was significantly decreased(p<0.01).Compared with NC group,the proportion of cells in the G0/G1 phase of the OE group was significantly decreased,and the proportion of cells in the S phase was significantly increased,and the difference was statistically significant(p<0.01).Flow cytometry was used to detect the effect of MIR4435-1HG on apoptosis of renal carcinoma cells:compared with the NC group,the proportion of apoptotic cells in the KD group was significantly increased,and the difference was statistically significant(p<0.01).The proportion of apoptotic cells in the OE group was significantly lower than that in the NC group,and the difference was statistically significant(p<0.01).Transwell assay showed:as for invasion,the number of cells in the OE group which penetrated the membrane was much larger than that in the NC group;the number of cells in the KD group which penetrated the membrane was much smaller than in the NC group(p<0.01).In terms of migration ability,the number of cells in the lower layer of transwell in the OE group was much larger than that in the NC group,and the number of cells in the lower layer of the transwell in the KD group was much smaller than that in the NC group(p<0.01).Conclusion:MIR4435-1HG knockdown and overexpression affect biological functions in renal carcinoma cells.MIR4435-1HG knockdown resulted in a decrease in cell proliferation,a decrease in cell clone formation,cell cycle arrest at G0/G1,induction of apoptosis in renal carcinoma cells,and a remarkable decrease in cell invasion and migration.In contrast,overexpression of MIR4435-1HG resulted in increased cell proliferation,increased cell colony formation,increased cell cycle S-phase ratio which suggests DNA replication,decreased renal carcinoma cell apoptosis,and significantly enhanced invasion and migration.In order to support the results of in vitro study,we will further confirm that in animal studies in vivo,and to explore the molecular mechanism of cell proliferation and apoptosis by studying interaction between MIR4435-1HG and proteins.Part ?:Mechanism of long non-coding RNA MIR4435-1HG affecting the biological behavior of renal cancer cellsObjective:To discover the molecular mechanism of MIR4435-1HG affecting the biological behavior of renal carcinoma cells.Methods:Probe was designed and fluorescence in situ hybridization was performed to detect the cellular localization of MIR4435-1HG in cells.The biotin RNA probe was labeled and the protein was isolated using RNA pulldown technology.The obtained protein was detected by mass spectrometry(LC-MS/MS).The protein of the cells was extracted.The results of the mass spectrometry were verified and other specific proteins were confirmed by western blot.Results:The results of fluorescence in situ hybridization showed that 18S and U6 localized cytoplasm and nucleus respectively.MIR4435-1HG was expressed in cytoplasm and nucleus,and the content in cytoplasm was relatively higher.RNA Pulldown identifies both the sense and antisense proteins followed by mass spectrometry,and comparison was made to find the unique proteins,ie,Muellerian-inhibiting factor(AMH),Pyruvate carboxylase(PC),Dermcidin(DCD),78 kDa glucose-regulated protein(GRP),Methylcrotonoyl-CoA carboxylase beta chain(MCCC2).In three cell lines,after MIR4435-1HG knockdown,as a cell cycle-associated protein BCL2L11 was found bo be increased.After MIR443 5-1HG overexpression,BCL2L11 was decreased than that of the control group.In addition to the fact that MCCC2 was not validated due to antibody purchase difficulties,we performed subsequent western blot assays for the remaining four RNA Pulldown proteins except MCCC2.After MIR4435-1HG knockdown,the expression level of PC were lower than in NC group.With MIR4435-1HG overexpression,the expression level of PC were higher than NC group.In addition,protein detection was performed with an important tumor-associated protein NICD1.The results showed that the expression level of NICD1 in MIR4435-1HG knockdown cells was lower than that in the control group.The expression level of NICD1 in cell lines overexpressing MIR4435-1HG was higher than that in the control group.Conclusion:MIR4435-1HG is mainly localized in the cytoplasm,suggesting that MIR4435-1HG plays a major role in post-transcriptional levels.Inhibition and overexpression of MIR4435-1MG can affect apoptosis,which is mainly achieved through BCL2L11 regulation.RNA pulldown and mass spectrometry showed MIR4435-1HG interacts mainly with the following proteins:AMH,PC,DCD,GRP,and MCCC2.The western blot results confirmed that there is a regulatory relationship between MIR4435-1HG and PC.In addition,MIR4435-1HG also regulates the expression level of NICD1.The finding of this study contributes to a more comprehensive explanation of the mechanism by which MIR4435-1HG plays a role in renal carcinoma cells.Part ?:Effects of long non-coding RNA MIR4435-1HG on proliferation of renal carcinoma cells in nude miceObjective:To discover the effects of long non-coding RNA MIR4435-1HG on kidney cancer in vivo in animals.Methods:SPF-class BALB/c nude mice were purchased from Shanghai SLAC Company.The 786-0 cell line transfected with knockdown virus or NC was collected.Cells were counted and prepared,and each nude mouse was injected subcutaneously.Each group had 8 nude mice.The tumor diameter was measured every 7 days for a total of 4 times.One month later,the nude mice were sacrificed and tumor tissues were taken and weighed.Results:The volume of subcutaneous xenografts in nude mice was found to change with time.There was no significant difference in the volume of subcutaneous xenografts between the two groups in the first week.The subcutaneous xenografts in the experimental group were smaller than the tumor size of the control group at the second,third and fourth weeks,p<0.001.Four weeks later,the nude mice were humanely sacrificed,the tumors of the nude mice were taken out,the size was measured,and the tumor was weighed.The results showed that the tumor volume of the experimental group was 116.8±56.75 mm3,and the volume of the tumor in the control group was 381.4±104.1 mm3.The tumor volume of the experimental group was significantly smaller than that of the control group,p<0.001.The tumor weight of the experimental group was 0.55±0.05g.The tumor weight of the control group was 1.17±0.04 g.The tumor weight of the experimental group was smaller than that of the control group,p<0.001.Conclusion:It is confirmed by subcutaneous transplantation of nude mice that the inhibition of MIR4435-1HG can restrict the growth of renal cancer in vivo,suggesting that MIR4435-1HG has certain prospects for the treatment of renal cancer.