The Mechanism Study Of LncCCAT1 Silence Promoting Apoptosis Of Renal Cell Carcinoma Cells By The Pathway Of Down-regulating Livin Protein Level

Background:Renal cell carcinoma(renal cell carcinoma,RCC)is the second most common urological tumor.The mortality of renal cancer has been increasing in the past 10 years,which seriously threatens the health of human beings.According to pathological results of cases samples,RCC can be divided into multiple subtypes.Among these subtypes,there are three subtypes accounting for about 90%of RCC,which are clear cell renal cell carcinoma(clear cell RCC),papillary renal cell carcinoma(papillary RCC)and chromophobe cell carcinoma(chromophobe RCC).Human clear cell RCC is the most common subtype and often occurs in the proximal tubule,accounting for about 75%of RCC.However,the molecular pathogenesis of RCC remains unclear.According to the existing researches,the molecular pathogenesis of RCC is an integrated biological process,which may involve the gradual accumulation of mutations,genomic instability,epigenetic changes and abnormal expression of protein-coding genes and non-coding genes.It is noted that the development of cancer genomics provides a lot of unprecedented information for exploring the mechanism of RCC.Among them,the role of non-coding RNAs(ncRNAs)has been widely concerned in recent years for its regulation function on gene expression,also it is involved in the research field of RCC.Genomics research showed that only about 1%to 2%of the human genes have protien encoding function,while more than 90%genes carry the non-protein coding information,which can synthesize ncRNAs and play other functions.According to the diversified functions,these ncRNAs can be broadly divided into two categories:housekeeping ncRNAs and regulatory ncRNAs.Housekeeping ncRNAs,such as transfer RNA,ribosomal RNA,etc.play the important roles in processes of gene transcription,post-transcription and translation.The regulatory ncRNAs have modulation function in processes of gene transcription and translation.Long non-coding RNAs(lncRNAs;more than 200nt)were discovered recently,which have regulating functions and can regulate the expression of protein-coding genes by the ways of cis-regulatory action and trans-regulatory role.Currently,studies have discovered a variety of IncRNAs involved in cancer occurrence and evolution,which played very important roles in the processes of cancer cell proliferation,apoptosis,migration and metastasis and other biological activities.The lncRNAs can promote proliferation or apoptosis of cancer cells in gene transcription stage and the post-transcription stage.Although the correlation research of lncRNAs with kidney disease is just in the early stage,the action of IncRNAs in kidney disease occurrence and development has been confirmed,and recently,researchers have observed the expressional changes of some IncRNAs under physiological or pathological conditions of kidney.Researchers have discovered a large number of IncRNAs with expression changes in renal cancer tissue by cDNA microarray and quantitative PCR techniques,moreover,the closed relevance between some IncRNAs and RCC occurrence has been confirmed.CCAT1(Colon cancer-associated transcript-1)is a newly discovered lncRNA,it locates on the 8q24.21 chromosome and contains 2628 base pairs.At first,some researchers found CCAT1 expression level higher in the colon cancer tissues than the para-carcinoma tissues,in the following study,the researchers discovered CCAT1 expression as oncogene in stomach,liver and gallbladder tissues.However,the role of CCATl in the process of RCC occurrence and development remains unclear.Infinite proliferation of cancer cells is a key aspect for cancer occurrence,inhibitor of apoptosis(IAP)have undeniable contributions in this process.Livin(also known as ML-IAP or KIAP)is a member of the IAP family.It plays a major role in a anti-apoptotic effect via inhibiting apoptotic signaling pathways.Livin is a 39kDa protein and consisted with a single BIR domain and a RINC finger domain.BIR domain is a zinc finger binding site consisting of about 70 amino acid residues,which can connect to the conservative cysteine-and histidine-rich residues,it is essential for anti-apoptotic activity of Livin.The upregulation of Livin in cancer cells is one of the important elements for cancer cell survival.The role of Livin in renal cell carcinoma has also been affirmed.Domestic and foreign literature searches showed that Livin gene expresses in embryonic tissues and malignant tumor tissues,but no in normal organ tissues.Livin gene distributes both in nucleus of tumor cells and the cytoplasm.Livin expression has a certain tissue specificity,the expression level is higher in lung cancer,stomach cancer,kidney cancer and bladder cancer than in other malignant tumor tissue.There are also studies confirmed that the the degree of malignancy was associated with the prognosis of renal cell carcinoma.Some domestic and foreign scholars have designed the interference sequence to block the expression of Livin in renal cell carcinoma,and it was found that the proliferation of renal cell carcinoma cells decreased and the percentage of apoptosis increased after the interference.Although the detailed mechanism of anti-tumor effect is not very clear,the Livin gene has become a hot topic for researchers,it is envisaged that it can be used as an effective target or bring new breakthroughs for the future treatment of malignant tumors and that,especially for renal carcinoma.We have long been engaged in clinical and basic research of renal cell carcinoma,we found in the early experiments that the CCATlrelative level is often high in highly malignant clinical specimens of renal cell carcinoma which has high Livin protein expression.So,we infer that there may be some regulatory mechanism or mechanism of interaction between the CCAT1 sequence and the Livin protein in kidney cancer,especially those highly malignant renal cell carcinoma.Recently,in addition to the their regulatory function in stages of gene transcription and post-transcription,lncRNAs can also directly combin to proteins and promote proteins degradation or stabilize proteins level,which contributed to the regulation of IncRNAs on tumorigenesis.So,whether there is correlation between CCAT1 and Livin protein in promoting tumorigenesis is the subject of our research.Therefore,the present study aims to investigate the correlation between IncRNA CCAT1 and Livin and their role in carcinogenesis from the following three research contents.Objective:1.To investigate the relevance between CCAT1 and renal cell carcinoma occurrence.Namely:whether CCAT1 can regulate the proliferation and apoptosis of RCC cells,whether it can affect the transplanted tumor growth of tumor-bearing mice.2.To investigate the expression of CCAT1 and Livin in renal cell carcinoma tissues and cell lines,the relationship between CCAT1 and Livin protein,and whether CCAT1 can regulate Livin protein level and the regulatory mechanism.3.To investigate whether the regulation of CCAT1 on Livin protein can carry out or participate in the regulation of CCAT1 on the proliferation and apoptosis of renal cell carcinoma cells.Methods:1.Real-time quantitative PCR was performed to determine the expression of CCAT1 in RCC tissue samples and RCC cell lines.2.Western blot was performed to detect the protein expression of Livin?Bcl-2?Bax?caspases3/7/9 in RCC tissue samples and RCC cell lines.3.MTT assay was performed to assess the cell activity and the apoptotic ratio of RCC cells.4.TUNEL and Flow cytometry assay were used to assess the apoptotic ratio of RCC cells.5.RCC cell lines were transfected with CCAT1 interference plasmid(named as si-CCATl)to explore the effect of si-CCAT1 on cell activity and apoptosis of RCC cells.RCC cell lines were transfected with si-CCATl and Livin overexpression plasmid(named as pcDNA3.1-Livin,hereinafter referred to as pcDNA-Livin)to explore the effect of pcDNA-Livin on the apoptosis promotion function of si-CCAT1.RCC cell lines were transfected with CCAT1 overexpression plasmid(named as pcDNA3.1-CCATl,hereinafter referred to as pcDNA-Livin)to explore the effect of pcDNA-CCAT1 on cell activity and apoptosis of RCC cells.RCC cell lines were transfected with pcDNA-CCAT1 and Livin interference plasmid(named as si-Livin)to explore the effect of si-Livin on the proliferation promotion function of pcDNA-CCAT1.6.The experiments of RNA pull-down and RNA-binding protein immunoprecipitation were performed to detect the Livin protein level combined by CCAT1 and CCAT1level combined by Livin protein and to investigate the relevance between CCAT1 and Livin protein.Western blot was performed to detect the Livin protein level after 786-0 cell line was treated with protein synthesis inhibitor cycloheximide(CHX)and proteasome inhibitor(MG132)respectively.After transfection of 786-0 cells by pcDNACCAT1,the 786-0 cells were treated subsequently with protein synthesis inhibitor(CHX)and proteasome inhibitor(MG132)respectively.Western blot was performed to detect the Livin protein level again.7.CCAT1-RNAi lentiviral recombinant vector was built and was transfected into RCC cell line to establish the stable transfected RCC line named 786-OLV-sh-CCAT1.The 786-OLV-sh-CCAT1 cells were transplanted subcutaneously into nude mice to observe the effect of CCAT1 silence on tumor growth.Results:1.Expression of CCAT1 and Livin protein in renal cancer tissues and RCC cells.Fluorescence quantitative PCR and western blot were performed to detect the expression levels of COATI and Livin protein in renal cancer tissues,para-carcinoma tissues and RCC cells.The results showed that CCAT1 expression and Livin protein expression in renal cancer tissues were significantly higher than in para-carcinoma tissues(p<0.001).The expression levels of COAT1 and Livin protein in 786-0 and Caki-1 cells were also significantly higher than in HK-2(p<0.001).2.The correlation analysis between CCAT1 expression level and clinical characteristic of RCC patients.The high CCAT1 expression(more than 3.916 times than para-carcinoma tissues)is closely related to tumor size and lymph node metastasis(P<0.05),but not the patient's gender,age,tumor grade,tumor stage,and distal metastasis(P>0.05).3.Effects of transfection of si-CCAT1 and pcDNA-CCAT1 on proliferation and apoptosis of human renal clear cell carcinoma cells.Compared with the si-control and MOCK groups,cell activity of si-CCAT1transfected group was significantly decreased and apoptotic cell ratio was significantly increased(The activity of si-CCAT1 transfected 786-0 cells was lower than that of the control groups at 48h,72h and 96h,the statistical difference was significant,P<0.001.The activity of si-CCAT1 transfected Caki-1 cells was lower than that of control group at 48h,72h and 96h,the statistical difference was significant,at 48h P<0.001,at 72h P=0.004,at 96h P=0.001.).Cell activity of pcDNA-CCATl transfected group was significantly increased and apoptotic cell ratio was significantly decreased(The activity of pcDNA-CCAT1 transfected 786-0 cells was higher than that of the control groups at 48h,72h and 96h,the statistical difference was significant,at 48h P=0.001,at 72h P=0.001,at 96h P=0.016.The activity of pcDNA-CCAT1 transfected Caki-1 cells was higher than that of control groups at 48h,72h and 96h,the statistical difference was significant,P<0.001).4.Effects of transfection of si-CCATl or pcDNA-CCAT1 on expression of Livin protein in RCC cells.si-CCAT1 promoted reduction significantly of Livin protein expression of the 786-0 cells and Caki-1 cells(compared with the control groups,P<0.001).pcDNA-CCAT1 promoted up-regulation significantly of Livin protein expression of the 786-0 cells and Caki-1 cells(compared with the control groups,P<0.001).5.pcDNA-Livin reverse the apoptosis promotion function of si-CCAT1(compared with si-CCAT1 group,P<0.001)and promote the proliferation of RCC cells.(compared with si-CCAT1 group,P<0.001).si-Livin reverse the proliferation promotion function of pcDNA-CCAT1(compared with pcDNA-CCAT1 group,P<0.001)and increase the apoptosis proportion of RCC cells(compared with pcDNA-CCAT1 group,P<0.001).6.CCAT1 promotes Livin protein stability in RCC cells by combining with it.RNA-pull-down experiments confirmed that CCAT1 can combine with Livin protein,and RNA-binding protein immunoprecipitation experiments confirmed that Livin protein can combine with CCAT1.Protein synthesis inhibitors(CHX)can inhibit Livin protein level.Proteasome inhibitor(MG 132)can upregulat Livin level.The Livin protein level was up-regulated in CCAT1 overexpressing 786-0 cells treated by CHX,but the Livin protein level was not affected in CCAT1 overexpressing 786-0 cells treated by MG 132.7.Effect of CCAT1 silence on transplanted tumor growth of tumor-bearing mice.In nude mice transplantation tumor mode,compared with 786-OMOCK and786-OLV-contro0 groups,the transplanted tumor growth of 786-OL-sh-CCAT1 group was inhibited.The statistical difference of transplanted tumor volume between 786-OL-sh-CCAT1 and control groups was significant 21 days after subcutaneous implantation(P<0.001).The average tumor weight of 786-OL-sh-CCAT1 group was 0.118�0.006g,the average weight of 786-OMOCK and 786-OLV-control groups were 0.259�0.015g and 0.261 �0.013g respectively and t test showed that the statistical difference was significant between the experimental group and the control groups.Compared with the control groups,the expression of Livin protein in 786-OLV-SH-CCAT1 group was significantly decreased(P<0.001).Compared with the control groups HE staining,786-OL-sh-CCAT1 group showed degeneration and necrosis in cancer cells:cell arrangement disorder and cytoplasmic swelling.Compared with the control groups,Livin protein and CD 10 protein immunohistochemical staining of 786-OL-sh-CCAT1 group were shallow,and the quantity of cells stained were less.Transmission electron microscopy showed that the tissue of 786-OL-sh-CCAT1 group had more apoptotic cells and apoptotic bodies than the tissues of control groups.Conclusion:1.In renal cancer tissues and cell lines,CCAT1 can regulate the proliferation and apoptosis of cancer cells,also CCAT1 can affect the transplanted tumor growth of tumor-bearing mice.2.In renal cancer tissues and cell lines,CCAT1 and Livin protein expression were significantly higher than that in para-carcinoma tissues and normal cells,the CCAT1 expression and Livin protein expression were positively correlated in renal cancer tissues.CCAT1 stabilizes the Livin protein level in cancer cells by combining with it.3.In renal cancer tissues and cell lines,the regulation of CCAT1 on Livin protein level contributes to the regulation of CCAT1 on the proliferation and apoptosis of RCC cells.