Study On The Function And Mechanism Of GRK6 Gene In Lung Adenocarcinoma

1.Background and Purpose:Lung cancer is the malignant tumor with the highest morbidity and mortality in the world.Lung adenocarcinoma has become the most common pathological subtype of lung cancer.In recent years,the treatment methods for lung adenocarcinoma include surgery,radiotherapy,chemotherapy,molecular targeted therapy,immunocheckpoint inhibitor therapy and other treatment measures,especially the latter two treatment measures have brought significantly prolonged overall survival benefit to lung cancer patients.Nevertheless,the survival prognosis of lung adenocarcinoma is poor.There are many reasons for its low survival rate,such as difficulty in early diagnosis,easy recurrence of cancer and high rate of early metastasis.The occurrence,development,metastasis and recurrence of lung adenocarcinoma are complicated,in which the inactivation of tumor suppressor genes and the activation of related oncogenes are involved in this process and play an important role.At present,the precise molecular mechanism of the occurrence and progression of lung adenocarcinoma is still unclear,so it is particularly urgent to explore new biomarkers of lung adenocarcinoma,which has important scientific significance for revealing the pathogenesis,prognosis and developing new therapeutic drugs of lung adenocarcinoma.GRK6,a member of the G protein-coupled kinase family,has been found to be associated with the occurrence,invasion and metastasis of numerous tumors in recent years.GRK6 has different and diverse functions in different types of malignant tumors,both promoting tumor growth and inhibiting tumor growth.However,the study on GRK6 gene in human lung adenocarcinoma has not been reported,so this study aims to explore the relationship between GRK6 and human lung adenocarcinoma.The purpose of this study was to clarify the expression of GRK6 gene in human lung adenocarcinoma and its relationship with clinicopathological parameters.To clarify the possible reasons for the down-regulation of GRK6 expression in lung adenocarcinoma and the regulatory mechanism of transcription factor C/EBPa on GRK6.To investigate the role of C/EBPa/GRK6 signaling pathway in the migration and invasion of lung adenocarcinoma cells,and to provide new ideas and methods for the prevention and treatment of lung adenocarcinoma.2.Methods:Using immunohistochemical method to determination of GRK6 in lung adenocarcinoma tissue microarray gene protein expression levels,protein immunoblot assay 18 cases of fresh specimens of lung adenocarcinoma tissues and adjacent carcinoma specimens of lung tissue protein expression,qPCR determination of 20 patients with fresh lung adenocarcinoma cancer tissues and matched adjacent to the carcinoma specimens GRK6 gene mRNA in the lung tissue.The relationship between expression level of GRK6 protein and related clinicopathological parameters and prognosis was statistically analyzed.Application of methylation specific PCR and bisulfite sequencing the PCR detection of 54 cases of lung adenocarcinoma in clinical specimens GRK6 gene promoter region methylation status,analysis of GRK6 promoter methylation status in its relationship with clinicopathological parameters and prognosis,and apply bisulfite sequencing the PCR detection of lung cancer cell line A549,A427,cell line H1299 and bronchial 16 HBE GRK6 in the promoter region methylation status,the further application of methylation transferase inhibitor treatment of lung cancer cells after observation of GRK6 expression level changes.Bioinformatics and chromatin immunoprecipitation were applied to explore and verify the transcription factor C/EBPa that may be involved in the regulation of GRK6 expression.Recombinant plasmids were constructed using wild-type and mutant GRK6 promoters,and luciferase reporter gene detection was performed to investigate the effect of possible binding sites of C/EBPa and GRK6 on GRK6 gene transcription.Chromatin immunoprecipitation was used to detect the binding of C/EBPa and GRK6 gene promoter in lung adenocarcinoma tissues and adjacent non-cancerous lung tissues.Lung cancer cell line A549 was demethylated with 5-aza-2 '-deoxycytidine,and the binding of C/EBPa and GRK6 promoter region was detected by chromatin immunoprecipitation.GRK6 expression was observed through silencing and overexpression of C/EBPa.The migration and invasion abilities of lung adenocarcinoma cells were observed by silencing and overexpression of GRK6.The expression levels of EMT-related markers E-cadherin and vimentin were observed by regulating the GRK6 expression levels of lung cancer cells A549 and H1299,and the levels of matrix metalloproteinases MMP2 and MMP7 were observed by overexpressing the GRK6 levels of lung cancer cells A549..3.Results:GRK6 gene expression level was significantly down-regulated in lung adenocarcinoma tissues,and GRK6 positive staining was mainly located in the cytoplasm of lung adenocarcinoma cells.The expression level of GRK6 was significantly correlated with the clinical staging and pathological grading of lung adenocarcinoma patients,but was not significantly correlated with the clinicopathological parameters such as gender,age,smoking history,tumor size and lymph node status.Cox proportional risk model multivariate analysis showed that GRK6 expression(P=0.004)and pathological grading(P<0.001)were independent prognostic indicators of patients' overall survival.Kaplan-meier survival analysis showed that the low expression of GRK6 was significantly correlated with the poor overall survival rate of patients with lung adenocarcinoma(P<0.001).MSP showed that among the 54 patients with lung adenocarcinoma,there were 42 patients with hypermethylation and partial methylation in GRK6 promoter region,and the methylation level was correlated with the degree of pathological differentiation(P=0.024),but there was no correlation between the methylation level of GRK6 promoter region and age,gender,smoking status,lymph node metastasis and tumor stage.Kaplan-meier survival analysis showed that there was a difference in OS among the three groups with different methylation levels(P=0.027),but there was no difference in OS between the non-methylated and partial methylated groups(P=0.492).BSP method detected that the methylation frequency of GRK6 gene promoter region CpG was 52±7.8%and 23 ± 6.5%respectively in lung adenocarcinoma tissues and adjacent non-tumor tissues,and the methylation level of GRK6 gene promoter region in lung adenocarcinoma tissues was significantly higher than that in adjacent non-tumor tissues.The mRNA level of GRK6 gene in lung cancer cell lines A549 and A427 was significantly lower than that in bronchial cell line 16HBE.The mRNA level of GRK6 gene in lung cancer cell line H1299 was no different from that in lung cancer cell line 16HBE.The methylation level of GRK6 promoter region in A549 cell lines was 63.9%,56.7%,and 15.6%,respectively,in A427 cell lines,H1299 cell lines,and 16HBE cell lines.The expression of GRK6 in A549 and A427 cells was up-regulated after the demethylation of 5-aza-2 '-deoxycytidine on lung cancer cells.Online prediction software and chromatin immunoprecipitation technology found three C/EBPa transcription factor binding sites in CpG island of GRK6 gene promoter region(S1:-793/-784 bp).S3:-394/-385 bp;S4:-198/-189 bp),the recombinant plasmid was constructed using the wild-type and mutant GRK6 promoter,and the luciferase reporter gene was detected.The results showed that the transcriptional activity was significantly reduced in the promoter region deletion binding sites S1,S3 or S4 groups,and the difference was statistically significant.Chromatin immunoprecipitation was used to compare the binding of C/EBPa and GRK6 gene promoter in lung adenocarcinoma tissues and adjacent non-cancerous tissues.After the immunoprecipitation reaction of C/EBPa antibody,the number of DNA fragments was amplified by different primers,and the results showed that the number of lung adenocarcinoma tissues was significantly lower than that of adjacent non-cancerous tissues.After the treatment of 5-aza-2 '-deoxycytidine on lung adenocarcinoma cell line A549,it was found that compared with the untreated control group,the binding sites S1,S3 and S4 of C/EBPa and GRK6 gene promoter region were significantly increased in lung cancer cells treated with 5-aza-2'-deoxycytidine.After silencing C/EBPa expression by siRNA and upregulation of C/EBPa expression by pcdna3.1-c/EBPa,protein western blot showed that the expression of C/EBPa in cells treated with C/EBPa siRNA was significantly decreased compared with the control group.On the contrary,the expression of C/EBPa in cells transfected with pcdna3.1-c/EBPa was significantly higher than that in the control group.In addition,inhibiting the expression of C/EBPa significantly down-regulated the expression of GRK6 protein,while promoting the expression of C/EBPa significantly up-regulated the expression of GRK6 protein.The expression of GRK6 was inhibited and promoted by the use of siRNA and overexpressed plasmids.After transfection with siRNA,the expression of GRK6 protein decreased significantly.On the contrary,the expression of GRK6 protein increased significantly after transfection with pcdna3.1-grk6.After transfection of pcdna3.1-grk6 into A549 cells,the expression of GRK6 protein was up-regulated.The migration test results showed that the migration ability of tumor cells was significantly inhibited,and the invasion test showed that the invasion ability of tumor cells was also significantly inhibited(P<0.05).On the contrary,when H1299 cells inhibited GRK6 expression by siRNA,the migration and invasion ability of tumor cells was significantly enhanced compared with the control group(P<0.05).After regulating the expression of GRK6 in lung cancer cell line A549,the expression changes of E-cadherin and vimentin were detected,and the overexpression of GRK6 led to up-regulation of E-cadherin and down-regulation of vimentin(P<0.05).On the contrary,in cells with down-regulated GRK6,E-cadherin expression decreased and vimentin expression increased(P<0.05).The levels of MMP2 and MMP7 in lung adenocarcinoma cell line A549 after the regulation of GRK6 expression were detected,and the levels of MMP2 and MMP7 were significantly inhibited by overexpression of GRK6(P<0.05).4.Conclusions:GRK6 gene is down-regulated in human lung adenocarcinoma,and the expression level of GRK6 gene is correlated with the degree of pathological differentiation and clinical staging of lung adenocarcinoma,which can be used as an independent prognostic indicator of lung adenocarcinoma.The down-regulation mechanism of GRK6 gene expression in lung adenocarcinoma is related to the hypermethylation state of this gene promoter region,which inhibits the binding of transcription factor C/EBPa to the gene.In lung adenocarcinoma,C/EBPa/GRK6 signaling pathway may be involved in tumor cell migration and invasion by promoting epithelial-mesenchymal transformation and increasing MMP2 and MMP7 levels of matrix metalloproteinases.