The Effects On The Biological Behaviors Of Renal Cell Carcinoma Of RECQ1 And Its Molecular Mechanism

Bacground Renal cell carcinoma(RCC)is a common malignant disease in urinary systyme,it derives from renal tubular epithelial cells and is highly heterogeneitive.Its incidence in adults accounts for about 2%-3% of malignant tumors.Renal cell carcinoma has a variety of pathological types,including renal clear cell carcinoma,papillary carcinoma,chromophobe carcinoma,of which renal clear cell carcinoma is the most common.The RECQ1(RECQL,RECQL1)helicase is the most abundant member of the RECQ family.Its main function is to promote the unwinding of DNA duplexes and the annealing of single-stranded DNA.RECQ1 also participates in DNA damage repair and maintenance of genome stability.At present,some studies have confirmed that silence of RECQ1 can inhibit biological behaviors such as proliferation,tumorigenesis,invasion,metastasis and epithelial-mesenchymal transition(EMT)of many kinds of malignant tumor cells.However,its expression in renal cell carcinoma and its relationship to the biological behaviors of renal cell carcinoma has not been reported yet.With the maturation of RNAi(RNAi)technique,it has been widely used in the field of molecular biology.Plasmids,si RNA or lentiviral vector can be transfected into cells to specific degradate a certain kind of m RNA,thus to down-regulated gene expression.The lentiviral vector is derived from retrovirus,which is widely used in cell experiments.After a series of transformations,the HIV-1 pathogenic sequence has been deleted,it has good safety and high infection efficiency,can carry longer RNA fragments and integrate the target sequence into the host's genome.After the Human Genome Project,omics studies such as transcriptomics,proteomics and glycomics have been rapidly developed.Based on the new generation of high-throughput sequencing technology,high-throughput RNA sequencing(RNA-seq)has been rapidly developed.Compared with the gene chip technology used in the past,RNA-seq eliminates the probe design and has the advantages of high signal-to-noise ratio,high resolution and large amount of data and provides an ideal platform for medical research.Mitogen-activated protein kinase(MAPK)signaling plays an important role in cell proliferation and differentiation.The MAPK pathway is activated by various extracellular stimuli and phosphorylation of MAPK occurs after cascade amplification of signal transduction including extracellular signal-regulated protein kinase(ERK),c-Jun-NH2 kinase(JNK)and p38 MAPK(p38).Studies have shown that the activation of the MAPK signaling pathway exists during the development,invasion,metastasis and angiogenesis of multiple human malignancies,including RCC.Enhanced ERK expression or activity has been detected in a variety of human tumors including breast cancer,glioblastoma,and primary tumor cells derived from kidney,colon and lung tissues.MAPK pathway activation and high expression of MKK1 and ERK2 also exist in clear cell renal cell carcinoma.Epithelial-mesenchymal transition is a key process for tumor cells to acquire metastatic potential.During EMT,epithelial cells lose their apical-basal polarity,and cell-cell junctions are destroyed,and eventually obtain typical mesenchymal features.E-cadherin and N-cadherin are key biomarkers that reflect the level of EMT.The decrease in E-cadherin expression is in balance with an increase in N-cadherin,indicating a decrease in cellular interactions and an increased ability of tumor cells to invade and migrate.A variety of signaling pathways are involved in the EMT process of tumor cells,such as transforming growth factor(TGF)-?,Wnt and nuclear factor(NF)-?B pathway,among which TGF-? is the most important EMT Related signal path.As a key transcription factor in the TGF-? pathway,Smad-3 binds to Smad-2 and then translocates into the nucleus via Smad-4 upon TGF-? receptor activation.Smad-3 regulates downstream genes and transcription factors such as ZEB1,ZEB2,Snail and Twist to exert EMT.In this study,we esplored the expression of RECQ1 in renal cell carcinoma by bioinformatics analysis,tissue microarray,Western Blot and immunofluorescence.A stable RECQ1 downregulation model was constructed in RCC cells ACHN and 786-O by constructing lentiviral vectors and infecting cells.The effect of RECQ1 downregulation on RCC cell transcriptome was revealed by RNA high-throughput sequencing.The impact of RECQ1 down-regulation on the biological behaviors of RCC cells were examined by CCK-8,colony formation,subcutaneous xenografts in nude mice,wound healing and Transwell assays.Finally,the KRAS-MAPK pathway and TGF-? pathway activities were detected to discover the underlying mechanism.Part 1 The Expression of RECQ1 in Renal Cell CarcinomaObjective: To investigate the expression of RECQ1 in renal cell carcinoma tissues and cellsMethods: The bioinformatics methods were applied to analyze the expression of RECQ1 m RNA in TCGA database.The relevant genes of RECQ1 were found.The gene ontology analysis and clustering analysis of the related genes were performed to predict the effect of RECQ1 in RCC.Immunohistochemical staining was performed on tissue microarray including RCC tissues and adjacent normal tissues,the expression of RECQ1 in RCC tissues and adjacent normal tissues was detected and statistically analyzed on the protein level.The expression of RECQ1 in RCC cells ACHN,786-O,769-P,A498 and renal tubular epithelial cells HK-2 was detected by Western Blot.The expression of RECQ1 in ACHN and 786-O cells was positioned by immunofluorescence.Results: Bioinformatics analysis showed that there were 1744 genes correlated with RECQ1(Pearson score> 0.4)in RCC.RECQ1 related genes were mainly associated with cell division,DNA replication,DNA transcription,DNA repair and small molecule GTPase activity.By immunohistochemical staining of tissue microarray,the number of RECQ1 positive cases in RCC group was 62 cases,the positive rate was 89.86%.In the adjacent normal tissues,the number of RECQ1 positive cases was 28 cases,the positive rate was 48.28%.The difference between the two groups was statistically significant(x2 = 22.95,p <0.001).In RCC tissues,46 cases(66.67%)expressed over ++.In the adjacent normal tissue,there were 19 cases with the expression level above ++,accounting for 32.76%.The difference was statistically significant(x2 = 14.50,p <0.001).By Western blot and immunofluorescence,we found that RECQ1 is expressed in the RCC cell lines ACHN,786-O,769-P,A498 and the tubular epithelial cell line HK-2,.The RECQ1 is located in the nucleus.RECQ1 was highly expressed in all four RCC cell lines compared to HK-2 cell line.Conclusion: 1.RECQ1 is highly expressed in RCC tissues at m RNA level compared to normal tissues adjacent to cancer.2.RECQ1 related genes in RCC are mainly associated with DNA replication,DNA transcription,DNA repair,small molecule GTPase activity.3.RECQ1 protein expression is higher in RCC tissues compared with paracancer normal tissues.4.RECQ1 is highly expressed in the RCC cell lines ACHN,786-O,769-P and A498 compared to the normal renal tubular epithelial cell line HK-2.RECQ1 is localized in the nucleus in RCC cells.Part 2 The Impact of RECQ1 Silencing on The Transcriptome of Renal Cell Carcinoma Objective: To construct a stable knockdown model of RECQ1 in RCC cells and to examine the effect of RECQ1 downregulation on transcriptome.Methods: Lentivirus vectors with sh-RECQ and unrelated control sequence were constructed by plasmid extraction and lentivirus packaging.The lentivirus-infected renal cell carcinoma cells 786-O and ACHN were seperated by G418 treatment to construct a stable RECQ1-silenced model.The knockdown efficiency of RECQ1 was tested by RT-q PCR and Western blot.RNA was extracted from NC and sh-RECQ1 transfected 786-O cells for high-throughput RNA sequencing.Results: Fluorescent proteins were stably expressed in both cell lines after lentivirus infection.RECQ1 m RNA and protein expression levels were significantly decreased in both cell lines.The sequencing data performed using the NC and sh-RECQ1 models established by 786-O cells was of good quality.In comparison with the NC group samples,there were 1279 genes whose expression levels were reduced by more than 50%(log2 ?-1)in the sh-RECQ1 group,and 1152 genes whose expression levels more than doubled(log2 ?1).GO analysis showed that the function of these down-regulated genes was mainly enriched in the binding of GTPases,regulation of small molecule GTPase activity,serine/threonine protein kinase activity and cell motility,etc.KEGG pathway analysis showed that RECQ1 downregulation mainly inhibited the signaling of MAPK,VEGF,TGF-? and m TOR pathways.Conclusion: 1.RECQ1 silenced models were constructed successfully and have provided a good foundation for the follow-up studies.2.RECQ1 down-regulation can cause changes of expression of a large amount of genes in the m RNA level.3.The down-regulation of RECQ1 can inhibit the proliferation,invasion and EMT-related signal transduction pathways,therefore RECQ1 could be a potential target for anti-tumor therapy.Part 3 The Effects of RECQ1 Silencing on The Biological Behaviors of Renal Cell Carcinoma and Its Molecular MechanismObjective: To investigate the effects of RECQ1 silencing on the biological behaviors of renal cell carcinoma and their molecular mechanisms.Methods: Stable cell lines with RECQ1 downregulation(sh-RECQ1)and unrelated sequence control(NC)were constructed using the renal cell carcinoma cell lines ACHN and 786-O.Cell viability was assayed by CCK-8 cell viability assay and colony formation assay.Migration assay was performed by wound healing assay and Transwell migration assay.Transwell assay was employed to detect cell invasiveness.Subcutaneous xenografts in nude mice was applied for the detection of Tumorigenicity.The effect of RECQ1 downregulation on cell epithelial-mesenchymal transition and the activity of MAPK and TGF-? pathway were detected by Western Blotting.Results: In both cell lines,the proliferation of sh-RECQ group was slower than that of NC group,and the difference of proliferation curve was statistically significant since 72 h.Knocking down of RECQ1 could significantly reduce the colony formation capacity of RCC cells with statistically significant differences.Through wound healing and Transwell migration assay,we found that down-regulation of RECQ1 in both RCC cell lines reduced the proportion of scratch healing and decreased the number of transmembrane cells,indicating that RECQ1 knockdown can inhibit migration ability.Through Transwell invasion assay,we found that RECQ1 knockdown resulted in a significant decrease in the number of transmembrane cells when coated with Matrigel,consists with a significant decrease in the expression of MMP-2/9,indicating that RECQ1 Knockdown can inhibit RCC cell invasiveness.In both RCC cell lines,the expression of K-RAS in sh-RECQ1 group was significantly decreased and the expression of RAF1,MEK1 and ERK1/2 was not significantly changed compared with NC group.However,p-MEK,ERK1/2 was obviously reduced,indicating that RECQ1 knockdown significantly inhibited the activity of KRAS-MAPK pathway.The expression of E-cadherin in the sh-RECQ group was significantly increased,the expression of N-cadherin and Vimentin was decreased,the expressions of EMT-related transcription factors Snail1 and ZEB1 were significantly decreased.The expression of Smad3 was not significantly changed while p-Smad3 expression decreased.The results showed that RECQ1 knockdown can inhibit the EMT of RCC cells,and this inhibition probably is achieved through the inhibition of TGF-? signaling pathway.Conclusion: 1.RECQ1 silencing can inhibit RCC cell proliferation and tumorigenicity.2.RECQ1 silencing can inhibit the invasion and migration of RCC cells.3.RECQ1 silencing can inhibit the KRAS-MAPK pathway activity.4.RECQ1 silencing can inhibit the epithelial-mesenchymal transition of RCC cells.5.RECQ1 silencing can inhibit the TGF-? pathway activity.