| Background In recent years,the pathogenesis of tumors is thought to be caused by mutations in some genes,that is,mutations in proto-oncogenes or expression of tumor suppressor genes is suppressed.At the same time,as the research on gene expression has gradually deepened,it has been found that translation of epigenetic control DNA and modification of histones have played a key role in regulating the expression of some proto-oncogenes and tumor suppressor genes.Among them,the role of G9 a is widely mentioned.G9 a belongs to the group of histone methyltransferases(HMTs),which is the lysine methyltransferase(H3K9)of histone ninth.In known studies,G9 a is involved in the occurrence and development of many cancers through H3K9.The main mechanism is through the methylation of histones in chromatin,which regulates the degree of histoneDNA binding,thereby reducing or silencing(modulating High or activated)expression of some key genes.In most cases,G9 a regulates gene expression by suppressing gene transcription,and these have been verified in vivo and in vitro in many types of tumor cells.In colorectal cancer,liver cancer,gastric cancer,pancreatic cancer,and ovarian cancer,G9 a directly inhibits the promoter region of certain tumor suppressor genes by methylating H3K9 to suppress gene expression.For example,histone methyltransferase G9 a promotes the development of liver cancer by suppressing the epigenetic silencing of the tumor gene RARRES3.Therefore,G9 a may be an important link in the development of cancer.However,the mechanism of action of G9 a in renal cancer has not been elucidated,so we aimed to study the mechanism of action of G9 a in renal cancer and evaluate the possibility of G9 a as a potential therapeutic target.In our study,we found that the expression of SPINK5 in renal cancer cell lines is inversely proportional to the expression of G9 a through bioinformatics.We speculated that SPINK5 may be a downstream target of G9 a,which was also obtained in the experiment.Confirmed.The serine protease inhibitor Kazal type 5(SPINK5)is located on human chromosome 5q32 and plays a role in anti-inflammatory and anti-microbial invasion.Studies have shown that SPINK5 plays a role in inhibiting tumorigenesis and development in a variety of cancers,such as esophageal,throat,bladder,lung,and cervical cancer.For example,SPINK5,a new tumor suppressor in esophageal cancer,is significantly reduced during the occurrence of esophageal cancer,and bioinformatics analysis and tissue arrangement of esophageal cancer are closely related to the pathological differentiation and lymph node metastasis of esophageal cancer,and SPINK5 Overexpression can inhibit Wnt / β-catenin signaling pathway and inhibit the proliferation,migration and invasion of esophageal cancer cells.In kidney cancer,SPINK5 also appears as a tumor suppressor gene.We tried to prove in vivo and in vitro experiments that G9 a can inhibit the expression of SPINK5 by binding to the SPINK5 promoter region and methylating the H3K9me2 site,thereby promoting kidney cancer.Cell proliferation,apoptosis,invasion and migration will further explore the mechanism of G9 a in renal cancer.Therefore,on the basis of previous research,this subject further studies how G9 a affects the biological behavior of renal cancer cells,and explores the role of the downstream target genes of G9 a to regulate the mechanism of tumorigenesis..In the first part,human kidney cancer cell lines(786-O,SN12 C,OSRC-2)were treated with UNC0638,a specific inhibitor of G9 a,and the effects of inhibited G9 a expression on the proliferation and apoptosis,migration and invasion of renal cancer cell lines were examined By means of CCK-8,clone formation,flow apoptotic staining,cell scratch test,Transwell cell invasion test and Western blot,etc.,a stable transfected renal cancer cell line was constructed at the same time,and the expression of G9 a on The effect of biological behavior of cancer cells;in the second part,the specific mechanism of G9 a gene regulating downstream target genes on the proliferation and migration,invasion and migration of renal cancer cells was further explored;in the third part,the subcutaneous migration of kidney cancer in nude mice was constructed In vivo experimental model of colonoma,nude mice were injected intraperitoneally with UNC0638 or inoculated with renal cell lines that knocked down G9 a expression after stable transfection.The effects of combined treatment on renal cell proliferation and apoptosis in nude mice were explored.Materials and methods1.We obtained kidney tumor tissues and adjacent tissues from 20 patients with renal cancer.The immunohistochemical staining of tissue sections was used to compare the expression of G9 a in human renal cancer cells and normal cells.Then through bioinformatics data mining,explore the expression of G9 a in human kidney cancer cell lines and clinical samples,and finally select human kidney cancer cell lines(786-O,SN12 C,OSRC-2)and human normal renal tubular epithelial cell lines HK-2.The expression was confirmed by Western blotting after protein extraction.High-expression G9 a was explored through bioinformatics analysis and Cancer Genome Atlas(TCGA)data obtained from the human protein atlas(https://www.proteinatlas.org/ENSG00000204371-EHMT2/pathology/renal+cancer#ihc)website Is related to poor prognosis of renal cancer;2.G9a-specific inhibitor UNC0638 is selective for a variety of epigenetic and nonepigenetic targets.Treatment of various cell lines with UNC0638 results in reduced levels of H3K9me2.We used UNC0638 to study the changes in biological behavior of renal cancer cell lines after G9 a function was inhibited.We selected two kidney cancer cell lines,786-O and SN12 C,and observed the proliferation of renal cancer cells after treatment with different concentrations and treatment time of inhibitor UNC0638 according to the CCK-8 test.The Grah Pad software calculated the two drugs for gastric cancer.The cell proliferation inhibition rate and the IC50 of the drug were finally determined to act on human kidney cancer cell lines 786-O24 h at final concentrations of 0,1.5 μM,3 μM,and 6 μM,and to affect human kidney cancer at final concentrations of 0,5 μM,10 μM,and 20 μM.The cell line SN12 C was tested by CCK-8,clone formation experiment,flow cytometry,scratch test,Transwell test and other methods to detect the effects of the inhibitor UNC0638 on the proliferation and apoptosis,migration and invasion of renal cancer cells.Proteins were extracted from the two cell lines treated with UNC0638 for 24 hours,and the expression changes of Bax,Bcl-2,H3K9me2,E-cadherin,N-cadherin,Vimentin and other proteins were detected by immunoblotting.3.Construct a stable cell line that knocks down G9 a.After knocking down G9 a,we performed CCK-8,clone formation experiment,flow cytometry,scratch test,transwell test and other methods to detect the cell biology of kidney cancer cell line 786-O and we performed western blot to detect changes in Bax,Bcl-2,H3K9me2,E-cadherin,Ncadherin,Vimentin and other protein expressions.4.In order to further study how G9 a regulates the biological behavior of renal cancer cells,we hypothesized that G9 a could epigenetically silence the expression of downstream genes to achieve its biological function.We identified five genes that may be downstream targets of G9 a.We found that only the expression levels of SPINK5 and p53 were up-regulated in kidney cancer cell lines that knocked down G9 a.Through the discovery of bioinformatics,we found in the ONCOMINE database that the expression of SPINK5 was negatively correlated with the expression of G9 a.G9a is upregulated in 6 renal cancer cell lines.In contrast,SPINK5 is down-regulated in these cell lines.Correlation analysis showed that the expression of G9 a was negatively correlated with the expression of SPINK5 in these renal cancer cell lines.We knocked down SPINK5 again in a stable G9 a cell line.We used CCK-8,scratch test,Transwell test and other methods to detect changes in the cell biological behavior of renal cancer cell line 786-O.At the same time,we used an immunoblotting method.The expression of E-cadherin,N-cadherin,Vimentin and other proteins in the cells was detected.Finally,the Ch IP test was used to prove the relationship between G9 a and SPINK5.5.Select the human kidney cancer 786-O cell line and the stable knockdown G9 a cell line,resuspend the cells in PBS to adjust the cell concentration to 4 × 107 / m L,and inoculate it subcutaneously in the back of nude mice(inoculation volume of 100 μL per nude mouse).After constructing a subcutaneous transplantation model of gastric cancer in nude mice,nude mice were randomly divided into groups.Two groups were implanted with 786-O and injected with normal saline and UNC0638.The two groups were implanted with negative control and test groups of knockdown G9 a renal cancer cell line.Observe nude mice.Status and growth of tumors were recorded.Nude mice were sacrificed by cervical vertebrae,subcutaneous transplanted tumors were separated and removed,tumor weights were weighed,tumor tissues were removed for immunochemical staining,and tumor growth curves and immunoblot methods were used to detect them.Bax,Bcl-2,H3K9me2,E-cadherin,N-cadherin,Vimentin expression.Results1.We obtained tumor tissues and adjacent tissues from 20 RCC patients and found that the expression of G9 a in renal cancer tissues was significantly higher than that in adjacent tissues.We extracted four pairs of proteins from renal cancer tissues and adjacent tissues.The results of immunohistochemistry remained consistent.Then,through bioinformatics data mining,G9 a was highly expressed in human kidney cancer cell lines and clinical samples from the human protein atlas,and its high expression was correlated with the poor prognosis of renal cancer(p <0.05).We further measured the expression level of G9 a in cell lines by Western blot experiments.We used the human renal tubular epithelial cell line hexokinase-2(HK-2)and three kidney cancer cell lines(ie 786-O,SN12 C,and OSRC-2).The results of Western blot analysis showed that the expression level in RCC cell lines was higher than that in HK-2 cells.Our results indicate that G9 a is generally highly expressed in renal cancer,and its high expression is related to the poor prognosis of RCC.2.By CCK-8 analysis,we demonstrated that UNC0638 significantly inhibited the growth of 786-O and SN12 C cells(Figure 2A).We set the drug concentrations of the two cell lines based on the results of the CCK-8 analysis,and finally determined that the human kidney cancer cell line 786-O24 h was acted on at a final concentration of 0,1.5 μM,3 μM,6 μM,and the final concentration was 0,5 μM,10 μM and 20 μM acted on the human kidney cancer cell line SN12 C.This experiment proved that as the concentration of the specific G9 a inhibitor UNC0638 increased,the proliferation ability of kidney cancer cells gradually weakened.We used Annexin-V-FITC / PI dual staining method to detect apoptosis by flow cytometry.The number of apoptotic 786-O and SN12 C cells gradually increased with increasing drug concentration.Similarly,colony formation experiments showed that as the drug concentration increased,the proliferation of 786-O and SN12 C cells was significantly inhibited,and the colonies formed were significantly reduced.According to the results of Western blot analysis,UNC0638 significantly inhibited H3K9me2 in 786-O and SN12 C cells.The expression of apoptotic gene Bax increased simultaneously with the increase of drug concentration,while the expression of apoptotic protection gene B-cell lymphoma 2(Bcl-2)decreased.This suggests that G9 a may promote tumor growth by inhibiting RCC cell apoptosis.We performed scratch and transwell experiments to explore the effects of G9 a on the migration and invasion of renal cancer cells.We found that the migration and invasion ability of 786-O cells was significantly inhibited after the inhibitor was added.Similar results were noted in SN12 C cells.Our results fully demonstrate that after inhibiting G9 a,the tumor’s ability to migrate and invade is significantly reduced.Our Western blot experiments showed that after treatment with inhibitors,the expression of E-cadherin increased significantly,while the expression of N-cadherin and vimentin decreased significantly.These findings indicate that the tumor’s epithelial-mesenchymal transition process was significantly inhibited after G9 a function was blocked.Therefore,G9 a may be closely related to the invasion and metastasis of renal cancer.3.We constructed a stable G9 a knockdown renal cancer cell line in 786-O to further explore the role of G9 a.After successfully knocking down G9 a,we observed a significant inhibition of cell proliferation after knocking down G9 a through the CCK-8test,and the apoptosis 786-was detected by flow cytometry using Annexin-V-FITC /PI dual staining method The number of O cells increased significantly compared with the control group.In addition,our clone formation experiments showed that the number of clones formed by 786-O cells after knocking down G9 a was significantly reduced compared with the control group,while the negative control group did not show significant changes compared with the blank control group.Similarly,we used scratch tests and Transwell invasion experiments to study the effect of reduced G9 a expression on the ability of renal cancer to migrate and invade.Our results indicate that the low expression of G9 a results in a significant reduction in renal cancer cell migration and invasion,which is consistent with results previously observed in experiments using specific inhibitors.Finally,Western blots confirmed these results.The expression of H3K9me2 was significantly decreased,the apoptosis-promoting gene Bax was upregulated,the apoptosis-inhibiting gene Bcl-2 was down-regulated,epithelialmesenchymal transition(EMT)promoted the gene down-regulation,and the EMT inhibitory gene was up-regulated.Based on these results,the loss of G9 a leads to a reduction in the carcinogenicity of kidney cancer.We hypothesized that the deletion of G9 a results in reduced methylation of the downstream target H3K9me2,thereby activating the expression of certain genes and ultimately leading to a reduction in carcinogenicity.4.We hypothesized that G9 a could epigenetically silence the expression of downstream genes to achieve its carcinogenicity.Therefore,we tried to explore the carcinogenic mechanism downstream of G9 a.Through literature review analysis,we identified five genes(SPINK5,ANKRD1,PTGS1,P53,RARRES3)that may be downstream targets of G9 a.We found that only SPINK5 and p53 were up-regulated in five genes after knocking down the expression of G9 a compared to the normal kidney cancer cell line786-O.In order to further analyze whether these two genes are downstream targets of G9 a,we used bioinformatics analysis to detect a correlation between G9 a and SPINK5.Through ONCOMINE data mining,G9 a was up-regulated in 6 cell lines(ACHN,786-O,RXF393,UO-31,TK-10,SN12C).In contrast,SPINK5 is down-regulated in these cell lines.Correlation analysis showed that the expression of G9 a was negatively correlated with the expression of SPINK5 in these renal cancer cell lines.Therefore,we assume that SPINK5 is one of the downstream target genes of G9 a.Using si RNA,we knocked down SPINK5 expression in a stable G9 a 786-O cell line.Through scratch test and Transwell chamber invasion experiment,we see that although the low expression of G9 a leads to a decrease in the migration and invasion ability of renal cancer cell lines,the migration and invasion ability of renal cancer cells is restored immediately after knocking down SPINK5.Western blot experiments also showed that the proliferation,migration,and invasion ability of 786-O cells expressing G9 a knockdown of SPINK5 was enhanced.Finally,in order to explore the relationship between G9 a and SPINK5,we designed a Ch IP test,G9 a can bind to the promoter region of SPINK5.The interaction between G9 a protein(H3K9me2 protein)and the downstream target SPINK5 promoter sequence was analyzed by Ch IP.Designed and synthesized specific primers that matched the SPINK5 promoter sequence.Analysis showed that G9 a binding levels and H3K9me2 levels in the SPINK5 promoter region were reduced after G9 a was silenced by sh RNA.Our salvage experiments and Ch IP experiments confirm our hypothesis that G9 a can enhance the carcinogenicity of renal cancer,which is achieved by the epigenetic silencing of the tumor suppressor gene SPINK5 by methylation of the H3K9me2 site in the promoter region.Interestingly,the expression levels of SPINK5 and p53 were up-regulated after G9 a inhibition.In remedial experiments,we found that p53 expression was down-regulated after knocking down both G9 a and SPINK5.This suggests that p53 may be a downstream target of SPINK5.5.After knocking down G9 a expression or using inhibitors to inhibit G9 a,xenograft tumors were significantly smaller than the control group.In our immunohistochemistry experiments,there were significant differences in the EMT index of in vitro xenografts.Immunohistochemical staining showed that the EMT index of G9 a showed significant changes in vitro.When G9 a is down-regulated or inhibited,the expression of Ncadherin is reduced,while the expression of E-cadherin is increased.Our Western blot analysis confirmed this finding.Our in vitro xenograft experiments show that G9 a can affect the proliferation and EMT process of renal cancer cells in vivo.This is consistent with the observations made in in vitro cell experiments.ConclusionG9a expression is generally up-regulated in renal cancer,while G9 a can promote the development of renal cancer in vitro and in vivo.Inhibiting or decreasing the expression of G9 a can reduce the proliferation,migration and invasion of renal cancer cells.In addition,we identified SPINK5 as one of the downstream target genes of G9 a.Therefore,targeting G9 a may be a new method for treating kidney cancer. |
PDF Full Text Request