| BackgroundRenal cancer is one of the most common malignant tumors of urinary system in adults.The latest national cancer survey data showed that the incidence rate of renal cell carcinoma accounted for about 5%of all cancer incidence rate,and was increasing year by year.Renal clear cell carcinoma(KIRC)is the main pathological type of renal cancer.With the popularization of physical examination and the progress of imaging technology,more and more renal cancer has been found in the early stage.However,about 30%of the patients had metastasis at the first diagnosis.At present,surgery is considered to be the most important and effective way to treat early renal cancer.However,20%-30%of tumor patients have recurrence or distant metastasis after surgery.Renal clear cell carcinoma is not sensitive to chemotherapy and radiotherapy,and the prognosis of patients with metastatic renal clear cell carcinoma is very poor,the five-year survival rate is less than 10%.Therefore,it is urgent and important to explore the mechanism of invasion and metastasis of renal clear cell carcinoma,to find the ideal tumor markers and effective treatment targets for the early diagnosis of KIRC patients,to improve the cure rate and overall survival rate of patients,and to improve the of KIRC patients.In recent years,more and more attention has been paid to the role of long non-coding RNA(lncRNA)in tumor.It has been found that lncRNAs can be used as a transcriptional regulator of oncogenes or tumor suppressor genes to participate in the proliferation,migration,invasion,metastasis,induction of drug resistance,immune escape and other processes of tumor.Through bioinformatics analysis,we screened out a new long non-coding RNA LINC00997,which was differentially expressed in renal clear cell carcinoma tissues and normal renal tissues adjacent to the cancer,and was closely related to the clinical stage and survival prognosis of the patients.In this study,we studied the effect of long non-coding RNA LINC00997 on the invasion and metastasis of renal clear cell carcinoma,and found that the expression level of long non-coding RNA LINC00997 was closely related to the invasion and metastasis of renal clear cell carcinoma.Epithelial mesenchymal transition(EMT)is a process in which epithelial cells change their morphology and behavior significantly when they differentiate into mesenchymal cells.In the EMT process of tumor cells,the expression of vimentin(VIM)and matrix metalloproteinases(MMPs)was enhanced,and the ability of anti apoptosis,degradation of extracellular matrix and cell migration were increased,which eventually led to tumor invasion and metastasis.EMT plays an important role in the invasion and metastasis of many malignant tumors,including renal cell carcinoma.We speculated that the long non-coding RNA LINC00997 might mediate the invasion and metastasis of renal cell carcinoma through EMT.S100A11 is one of the important cell regulatory factors in S100 protein family.It can participate in tumor proliferation,invasion,metastasis,immune escape and other processes,and is related to the EMT of glioblastoma,intrahepatic cholangiocarcinoma and other tumors.Will S100A11 be the target gene of long non-coding RNA LINC00997 mediated EMT?To test our hypothesis,we first predicted the expression of long non-coding RNA LINC00997 and S100A11,EMT related molecules(VIM,MMP2 and MMP7)through bioinformatics,and then designed a series of RNA interference experiments to confirm its expression and biological function in renal clear cell carcinoma cells.In order to further study the molecular mechanism of long non-coding RNA LINC00997 regulating S100A11,we verified the targeting relationship between long non-coding RNA LINC00997 and S100A11 promoter by bioinformatics analysis and dual Luciferase Report experiment,and confirmed that STAT3 is the transcription factor of S100A11.Then,RNA binding protein immunoprecipitation(RIP)confirmed that long non-coding RNA LINC00997 can bind STAT3.RNA interference experiment confirmed that the target gene of long non-coding RNA LINC00997 and transcription factor STAT3 was S100A11.Finally,we revealed the molecular mechanism of LINC00997 regulating the invasion and metastasis of renal clear cell carcinoma cells,and provided a potential tumor marker and drug treatment target for kidney renal clear cell carcinoma.Part I Expression and clinical significance of long noncoding RNA LINC00997 in kidney renal clear cell carcinomaAimVerification the expression of long noncoding RNA LINC00997 in primary renal clear cell carcinoma and normal renal tissues adjacent to the carcinoma,as well as in human renal clear cell carcinoma cell line 786-O and human renal tubular epithelial cell line HKC.The relationship between LINC00997 and distant metastasis,clinical stage and survival prognosis of patients was analyzed,so as to further study the function and regulatory mechanism of long noncoding RNA LINC00997 in kidney renal clear cell carcinoma.Method1.Through analyzing the GEPIA RNA-Seq datasets,we expore the expression level of long non-coding RNA LINC00997,and its relationship with kidney renal clear cell carcinoma.2.The Clinicopathological datas of 18 patients with renal clear cell carcinoma who underwent radical nephrectomy from June 2017 to December 2018 were collected,including distant metastasis and TNM stage.All the included specimens were reconfirmed by the pathology experts of the First Affiliated Hospital of Zhengzhou University.All experimental materials,including the acquisition of tumor and tumor adjacent tissue after operation,and further experiments were approved by the ethics committee of the First Affiliated Hospital of Zhengzhou University.The expression of linc00997 in renal clear cell carcinoma and matched normal tissues was detected by qRT-PCR.3.The expression of LINC00997 in 786-O cell line and HKC cell line was explored.Results1.Through analyzing the GEPIA RNA-Seq datasets,results demonstrated that LINC00997 is highly expressed in multiple cancers compared to normal tissues,including in KIRC,kidney renal papillary cell carcinoma(KIRP),prostate adenocarcinoma(PAAD)and so on.The expression of long noncoding RNA LINC00997 was up-regulated in stage ? and ? renal clear cell carcinoma.Kaplan-Meier survival curve analysis showed that overall survival(OS)and disease-free survival(DFS)rates of patients with high expression of long-chain non coding RNA linc00997 were significantly lower than those of patients with low LINC00997 expression(Logrank P=0.0062;Logrank P=1.8e-06).2.The expression of LINC00997 in the tumor tissue was significantly higher than that in the adjacent normal renal tissue,and the expression of LINC00997 in the patients with distant metastasis was significantly higher than that in the patients without metastasis,and the expression of LINC00997 in the patients of stage ? and? was significantly higher than that in the patients of stage ? and ?(P<0.05).3.The expression of LINC00997 in 786-O cell line was significantly higher than that in HKC cell line(P<0.01)ConclusionLINC00997 is highly expressed in kidney renal clear cell carcinoma and 786-O cell line.LINC00997 has a significant correlation with distant metastasis,clinical stage and survival prognostic in KIRC.The results of this part lay a foundation for further study of the role of long noncoding RNA LINC00997 in renal clear cell carcinoma,and also lay a foundation for further exploration of the mechanism of long noncoding RNA LINC00997 regulating the invasion and metastasis of renal clear cell carcinoma.Part ? Long noncoding RNA LINC00997 regulates the biologicalfunction of kidney renal clear cell carcinomaAim:In order to further study the role of LINC00997 in the invasion,metastasis and epithelial-mesenchymal transition of kidney renal clear cell carcinoma,we designed a series of RNA interference experiments to verify the expression relationship and related biological functions of long non-coding RNA LINC00997,S100A11 and EMT related molecules(VIM,MMP2 and MMP7)in this study.It established the foundation for further revealing the molecular mechanism of Ion non-coding RNA LINC00997 regulating kidney renal clear cell carcinomaMethod1.Through analyzing the GEPIA RNA-Seq datasets,we expored the relationship between long non-coding RNA LINC00997 and S100A11,and the expression level of S100A11 in renal clear cell carcinoma,and its relationship with clinical stage and survival prognosis of renal clear cell carcinoma.2.The expression of S100A11 was detected by qRT-PCR in 18 cases of renal clear cell carcinoma and matched normal tissues.3.Targeted siRNA silenced the expression of long non-coding RNA LINC00997 and S100A11 in human renal clear cell carcinoma cell line 786-O.We evaluated scratch repair ability and cell migration ability of 786-O cells by wound healing assay and Transwell migration test.4.Furtherly,RNA-Seq datasets from GEPIA were utilized to analyze the expression and association of LINC00997,S100A11,VIM,MMP2,and MMP7 with KIRC.5.Through silencing the expression of long non-coding RNA LINC00997 and S100A11 separetely,the expression of VEM,MMP2 and MMP7 were detected by qRT-PCR.Results1.Through analyzing the GEPIA RNA-Seq datasets,we found that LINC00997 was positive correlated with S100A11 in KIRC.S100A11 was highly expressed in multiple cancers.S100A11 was up-regulated in stage ? and ?.Kaplan Meier survival curve analysis showed that overall survival(OS)and disease-free survival(DFS)rates in highly expressed S100A11 KIRC patients were decreased compared to patients with low LINC00997 expression(Logrank P=1.1e-05;Logrank P=4e-04).2.Compared to matched normal tumor adjacent tissues,a highly expressed S100A11 was detected in primary kidney renal clear cell carcinoma.And that S100A11 expression in KIRC metastases was higher than in non-metastatic tumors.3.A wound healing assay showed that inhibiting LINC00997 or S100A11 decreased migration in 786-O cells.Transwell assays were also performed,demonstrating that interfering with LINC00997 or S100A11 expression inhibits migration of 786-O cells(both P<0.05).4.The GEPIA RNA-Seq datasets demonstrated that there were positive correlation of LINC00997,S100A11 and VIM,MMP2,MMP7 in KIRC.5.Silencing the expression of LINC00997 or S100A11 inhibits EMT associated molecular expression of VIM,MMP2 and MMP7(all P<0.05).Conclusion1.There was close correlation between the expression of long non-coding RNA LINC00997 and S100A11 in KIRC.2.S100A11 is highly expressed in renal clear cell carcinoma and has significant correlation with distant metastasis,clinical stage and survival prognosis.3.Long non-coding RNA LINC00997 and S100A11 could regulate the migration with 786-O cell line.4.Long non-coding RNA LINC00997 and S100A11 could regulate expression of VIM,MMP2,and MMP7..Part III The molecular mechanism of long non-coding RNA LINC00997 regulating the metastasis of kidney renal clear cell carcinomaAimThe aim of this study was to explore the targeted relationship between long non-coding RNA LINC00997 and S100A11,and to reveal the specific molecular mechanism of long non-coding RNA LINC00997 which regulate the invasion and metastasis of kidney renal clear cell carcinoma.Method1.Bioinformatics prediction:we retrieved the relationship between long non-coding RNA LINC00997 and S100A11 based on analysis using the LncTar database.Additionally,we explore the corelation between S100A11 promotor and STAT3 by using the JASPAR and hTFtarget database.Further more,potential relationship between LINC00997 and STAT3 protein was predicted by the LNCPRO database.2.The luciferase reporter gene plasmid of S100A11 promoter was constructed and transfected into HEK293 cells with si-LINC00997 or negative control respectively.We explore luciferase activity in si-LINC00997 group and negative control group by using dual Luciferase Report gene assay.3.Three luciferase reporter gene plasmids of S100A11 promoter were constructed in this study,including S100A11-pro-wt,S100A11-pro-1 mut and S100A11-pro-2mut.These luciferase plasmids and si-STAT3 or negative control were transfected into HEK293 cells.Luciferase activity of si-STAT3 group and negative control group was detected by dual luciferase reporter gene assay.4.The interaction between long non-coding RNA LINC00997 and STAT3 was detected by RNA binding protein immunoprecipitation(RIP).5.After silencing the expression of long non-coding RNA LINC00997 and STAT3 by siRNA,the expression of S100A11 in 786-O cell line was detected by qRT-PCRResults1.Long non-coding RNA LINC00997 is predicted to combine with the S100A11 promoter by usring LncTar database.The S100A11 promoter has two potential binding sites with the STAT3 protein based on analysis using the JASPAR and hTFtarget database.LINC00997 has the potential to combine with the STAT3 protein,as predicted by the LNCPRO database.2.Silencing long non-coding RNA LINC00997 significantly reduced the luciferase activity of S100A11 promoter,the difference was statistically significant.(P<0.05)3.Silencing STAT3 significantly inhibited the luciferase activity of S100A11 promoter wild type and type ? mutant(P<0.05),but there was no significant difference between S100A11 promoter type ? mutant and control group.4.Long non-coding RNA LINC00997 has the potential to combine with the STAT3 protein.5.Silencing long non-coding RNA LINC00997 or STAT3 resulted in reduced S100A11 expression in 786-O cells.Conclusion1.Long non-coding RNA LINC00997 could regulate S100A11 expression.2.STAT3 could combine with the promoter of S100A11 and promote the transcription of S100A11.3.Long non-coding RNA LINC00997 has the potential to combine with the STAT3 protein.4.Long non-coding RNA LINC00997 could regulate S100A11 expression by binding with STAT3 protein. |
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