| Renal cell carcinoma(RCC)is a common type of malignant tumor in urology,and its incidence is only second to bladder cancer and prostate cancer,ranking third in urological malignant tumor.In recent years,with the development of imaging technology and people’s increasing attention to regular physical examination,the incidence of RCC has been increasing year by year.According to the statistics,more than 70,000 new RCC patients occurred in the United States in 2021.Clear cell renal cell carcinoma(ccRCC)is the most common histological type of RCC,accounting for about 70%of all types.Currently,patients with early or localized ccRCC can achieve long-term survival after surgical treatment.However,for patients with advanced or metastatic ccRCC,the prognosis is often poor due to insensitivity to radiotherapy and chemotherapy.With the continuous progress of molecular biology technology,targeted drugs gradually come into people’s vision,and provide a glimmer of hope for the treatment of such patients.Despite this,many patients still have unsatisfactory outcomes.Therefore,finding new and more effective therapeutic targets for ccRCC is one of the key problems to be solved urgently.Whole genome sequencing technology has made great contributions to human health.With the rapid development of this technology,researchers found that only about 2%of human genes are eventually translated into protein,but the majority of human genes are not translated into protein,which is the non-coding RNA.The length of Long noncoding RNA(lncRNA)is generally less than 200nt,and most of them do not have the function of coding protein.Although lncRNAs cannot encode proteins,it can affect biological functions of the human body in other ways,such as gene transcription,post-transcriptional regulation,translation and post-translational modification,which are closely related to the occurrence and progression of human malignant tumors.Studies have shown that LINC01116 is abnormally expressed in a variety of malignant tumors,including osteosarcoma,glioma,gastric cancer and nasopharyngeal cancer,and participates in the regulation of the occurrence and progression of these tumors.Although LINC01116 has been shown to play an important role in a variety of tumors,its role in ccRCC remains unclear.In the early stage of this study,differentially expressed lncRNAs in ccRCC tissues and normal kidney tissues were conducted through GEPIA database,and LINC01116 was found to be highly expressed in ccRCC tissues.We verified the expression level of LINC01116 in ccRCC cell lines and ccRCC tissues,and analyzed the relationship between LINC01116 expression level and clinicopathological features and prognosis of patients.Subsequently,we verified the subcellular localization of LINC01116 in ccRCC cell lines by nuclear cytoplasmic separation assay.Through a series of in vitro and in vivo experiments,we preliminarily studied the effects of LINC01116 on the biological functions of ccRCC cells.In order to clarify the molecular biological mechanism of LINC01116 regulating the progression of ccRCC,we predicted the target molecule of LINC01116 through bioinformatics,then verified the predicted results at the RNA and protein levels,and further verified the predicted results by dual luciferase reporter gene assay.Finally,we further verified the molecular biological mechanism of LINC01116 regulating the progression of ccRCC through a series of rescue experiments,thus providing new ideas for clinical targeted treatment of ccRCC.Part Ⅰ:Expression of LINC01116 in ccRCC and its clinical significanceObjective:The abnormal expression of lncRNAs in ccRCC was screened,and the expression level of LINC01116 in ccRCC cell lines and ccRCC tissues was verified,and the relationship between the expression level and clinicopathological features and prognosis of patients was analyzed.Methods:LncRNA abnormally expressed in ccRCC were initially screened by the GEPIA database.The expression level of LINC01116 in ccRCC cell line and ccRCC tissue was verified by RT-qPCR.The correlation between LINC01116 expression level in ccRCC tissues and clinicopathology of ccRCC patients was analyzed.Kaplan-meier survival analysis was used to draw the patient survival curve.The subcellular localization of LINC01116 in ccRCC cells was verified by nucleo-cytoplasmic separation assay.Results:Analysis results of GEPIA database showed that the expression of LINC01116 in ccRCC tissues was higher than that in normal kidney tissues,and the difference was statistically significant.RT-qPCR results showed that the expression of LINC01116 in all ccRCC cell lines was higher than that in normal renal tubular epithelial cells HK-2,and the difference was statistically significant.The expression of LINC01116 in ccRCC tissues was higher than that in adjacent paired tissues,and the difference was statistically significant.According to the median expression level of LINC01116 in ccRCC tissues,LINC01116 was divided into high expression group and low expression group,and the clinicopathological characteristics and prognosis of patients in the two groups were compared.It was found that high expression of LINC01116 was associated with larger tumor and higher clinical stage.Survival analysis showed that high expression of LINC01116 was associated with reduced overall survival and disease-free survival.The results of nucleo-cytoplasmic separation showed that LINC01116 was mainly located in the cytoplasm of ccRCC cell lines.Conclusion:LINC01116 is highly expressed in ccRCC and is associated with poor clinicopathological features and poor patient outcomes.Part Ⅱ:Effects of LINC01116 on biological behavior of ccRCC cells in vitro and in vivoObjective:To investigate the effects of LINC01116 on biological functions of ccRCC cell proliferation,invasion,migration and cell cycle in vitro and in vivo.Methods:SiRNA or sh-RNA was transfected into ccRCC cells to interfere with LINC01116 expression,and the transfection efficiency was measured by RT-qPCR.CCK8 assay was used to detect the effects of LINC01116 on ccRCC cell proliferation,Transwell migration and invasion assay was used to detect the effects of LINC01116 on ccRCC cell migration and invasion,and flow cell cycle assay was used to detect the effects of LINC01116 on ccRCC cell cycle.Western blot assay was used to detect the effect of LINC01116 on epithelial-mesenchymal transition(EMT).The effect of LINC01116 on tumorigenesis of ccRCC cells in vivo was detected by subcutaneous tumor bearing experiment in nude mice,and the effect of LINC01116 on proliferation activity of ccRCC cells was detected by immunohistochemistry.Results:The RT-qPCR results showed that the transfection of siRNA or sh-RNA could significantly reduced the expression of LINC01116 in ccRCC cells.CCK8 results showed that interfering the expression of LINC01116 could weaken the proliferation ability of ccRCC cells.Transwell migration assay showed that interfering with LINC01116 expression could reduce the migration ability of ccRCC cells.Transwell invasion assay showed that interfering with LINC01116 expression could reduce the invasion ability of ccRCC cells.The results of the flow-cell-cycle experiments showed that the downregulation of LINC01116 expression arrested the cell-cycle of ccRCC cells in the G0/G1 phase.The Western blot results showed that downregulation of LINC01116 expression increased E-cadherin expression in EMT-associated proteins and decreased N-cadherin and Vimentin expression.Tumorigenicity of ccRCC cells was inhibited by down-regulation of LINC01116 expression in nude mice.Immunohistochemical results showed that downregulation of LINC01116 could reduce the proliferation activity of ccRCC cells.Conclusion:Silencing the expression of LINC01116 weakened the proliferation,invasion,migration and EMT abilities of ccRCC cells in vitro,and blocked the cell cycle.Silencing of LINC01116 expression decreased the tumorigenic ability and proliferative activity of ccRCC cells in vivo.Part Ⅲ:LINC01116 promotes the malignant progression of ccRCC by inhibiting the expression of miR-766-3pObjective:To investigate the targeted miRNA of LINC01116 in ccRCC cells and its mechanism.Methods:The downstream target molecules of LINC01116 were predicted by bioinformatics.The predicted results were verified by RT-qPCR,and the expression of miR-766-3p in ccRCC cells was detected.The expression of miR-766-3p after transfection with miR-766-3p inhibitor or miR-766-3p mimic in ccRCC cells was detected by RT-qPCR.The binding relationship between LINC01116 and miR-766-3p was verified by double luciferase reporter gene assay.A series of response experiments including CCK8 experiment,Transwell migration experiment and Transwell invasion experiment were conducted to verify whether the expression of miR-766-3p can partially restore the inhibitory effect of silencing LINC01116 on the malignant phenotype of ccRCC cells.Results:The bioinformatics prediction results showed that miR-766-3p may be one of the downstream target molecules of LINC01116 in ccRCC cells.The RT-qPCR results showed that miR-766-3p was low expressed in ccRCC cells,and its expression level increased with the decrease of LINC01116 expression level.Transfection of ccRCC cells with miR-766-3p inhibitor or miR-766-3p mimic could decrease and increase the expression level of miR-766-3p respectively.The results of the double luciferase reporter gene assay showed that there was a binding relationship between miR-766-3p and LINC01116.Inhibition of miR-766-3p expression can partially recover the inhibitory effect of LINC01116 on malignant phenotype of ccRCC cells.Conclusion:There is a binding relationship between LINC01116 and miR-766-3p,which promotes the malignant progression of ccRCC by inhibiting the expression of miR-766-3p.Part Ⅳ:LINC01116 promotes malignant progression of ccRCC via miR-766-3p/DDX11 signalling pathwayObjective:To screen the downstream target molecules of LINC01116/miR-766-3p axis in ccRCC cells and explore the specific mechanism of action.Methods:The downstream target molecules of miR-766-3p were predicted by bioinformatics.The prediction results were verified by RT-qPCR,and the expression of DDX11 in ccRCC cells was detected.The expression of DDX11 after transfection of miR-766-3p inhibitor into ccRCC cells was detected by RT-qPCR.The binding relationship between miR-766-3p and DDX11 was verified by double luciferase reporter gene assay.The expression of DDX11 after co-transfection of SI-LINC01116 and miR-766-3p inhibitor into ccRCC cells was detected by RT-qPCR and Western blot.Results:The bioinformatics prediction results showed that DDX11 might be one of the downstream target molecules of miR-766-3p in ccRCC cells.RT-qPCR results showed that DDX11 was highly expressed in ccRCC cells,and its expression level increased with the decrease of miR-766-3p expression level.The results of dual luciferase reporter gene assay showed that DDX11 had a direct binding site to miR-766-3p.RT-qPCR and Western blot results showed that co-transfection of Si-LINC01116 and miR-766-3p inhibitor weakened the promoting effect of RT-qPCR-766-3p inhibitor on DDX11 expression.Conclusion:There is a binding relationship between DDX11 and miR-766-3p,and LINC01116 can upregulate DDX11 expression by inhibiting miR-766-3p,thus promoting the malignant progression of ccRCC. |
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