| Background and Objective:Hepatocellular carcinoma（HCC）is one of the most common and highly malignant human malignant tumors in the world.The International Agency for Research on Cancer reports that there are about 800000 new cases and 700000 deaths every year.Tumor proliferation is a complex process,and many internal characteristics and external microenvironmental factors affect the proliferation of hepatoma cells[3].However,the potential molecular mechanism of mediating the proliferation of HCC is still unclear to a large extent.GRP78 is the core regulator of unfolded protein response（UPR）,which plays an important role in cell adaptation and survival under stress[4].Many studies have proved that GRP78 is highly expressed in a variety of tumors and has the potential to be used as a biomarker and therapeutic target[5-6].Our previous study also found that GRP78 is highly expressed in hepatocellular carcinoma,and the high expression of GRP78 is closely related to the occurrence and development of GRP78 is the core regulator of unfolded protein response[7-8].The above studies show that GRP78 can affect the occurrence and development of tumors,and GRP78 may be a potential target for regulating tumor proliferation.TGF-βsignaling pathway has been proved to play a very important and extensive role in growth and development,tumorigenesis and development[9-10].More and more evidence shows that TGF-βsignaling pathway promotes the survival and proliferation of cancer cells after the formation of hepatocellular carcinoma[11-13].Smad Anchor For Receptor Activation（SARA）is considered to be the key protein regulating TGF-βsignaling pathway.Related studies have shown that SARA can directly interact with Smad2/3 and TGF-βreceptor complex and directly phosphorylate Smad2/3[14-15].Phosphorylated Smad2 and Smad3 form a trimer with Smad4,which then translocates to the nucleus and binds to transcription factors to regulate gene transcription and promote tumor development[16-17].However,at present,the regulation mode of SARA and its role in hepatocellular carcinoma are not clear.Whether GRP78 affects TGF-βsignal pathway by regulating SARA needs to be further explored.Methods:1.HCCLM3 cells stably interfering with GRP78 were constructed.shGRP78-HCCLM3 cells and NC-HCCLM3 were collected for RNA-seq.Bioinformatics was used to analyze differentially expressed genes to explore the biological processes and molecular functions related to GRP78 in hepatoma cells.2.Furthermore,by analyzing the expression level of GRP78 and SARA in patients with HCC in TCGA and ICGC database,the correlation of GRP78 and SARA expression was analyzed,and its clinical significance was explored.3.To explore the expression of GRP78 and SARA in different hepatoma cell lines,select one cell line with relatively high expression of GRP78 protein and one cell line with relatively low expression of GRP78 protein,then interfere with the overexpression of GRP78,in cells with high expression of GRP78 and overexpression of GRP78,in cells with low expression of GRP78 to detect the changes of mRNA and protein of GRP78 and SARA,and observe the changes of proliferation ability of hepatoma cells by CCK8 and EdU experiments.4.The recovery test was used to detect the upstream and downstream relationship between GRP78 and SARA.Interfere with GRP78 and increase the expression of SARA in hepatoma cells with high expression of GRP78,correspondingly,overexpression of GRP78 and interference with the expression of SARA in hepatoma cells with low expression of GRP78.The changes of mRNA and protein of GRP78 and SARA were observed,and the proliferation ability of hepatoma cells was observed by EdU assay.At the same time,the changes of Smad2/3 and P-Smad2/3 proteins were observed after interfering with or overexpressing GRP78.Results:1.RNA-seq of shGRP78-HCCLM3 cells and NC-HCCLM3 cells showed that174 genes were significantly up-regulated and 133 genes were significantly down-regulated after interfering with GRP78 expression.Through Metascape analysis,it was found that the biological processes and molecular functions such as TGF-βsignaling pathway and cell response to growth factor stimulation were significantly affected,and IPA analysis also found that TGF-βsignaling pathway was significantly inhibited after interfering with GRP78 expression.Further analysis showed that the expression level of SARA,the key protein of TGF-βsignaling pathway,decreased significantly after interfering with GRP78.2.Comprehensive RNA-seq and transcriptome data analysis of HCC patients in public database TCGA and ICGC showed that the expression of GRP78 and SARA increased significantly in HCC tissues.The correlation coefficient between GRP78and SARA expression in TCGA database was 0.35 and the P-Value=9.6x10-14;the correlation coefficient between GRP78 and SARA expression in ICGC database was0.38 and P-Value=2.2x10-16.TCGA database showed that the expression level of GRP78 was positively correlated with the shorter overall survival time of patients with HCC,and the expression level of SARA was significantly positively correlated with the shorter overall survival time and progression-free interval of patients with HCC.3.The results of cell screening showed that the expression of GRP78 and SARA in HCCLM3 was higher,the expression of GRP78 and SARA in SMMC7721 was low.Western blotting and q RT-PCR experiments showed that the silencing of GRP78could decrease the expression of SARA in HCCLM3 cells,while over-expression of GRP78 could increase the expression of SARA in SMMC7721 cells.The result of cellular immunofluorescence experiment is the same.4.The results of CCK8 test and EdU proliferation test showed that interfering with GRP78 inhibited the proliferation of HCC cells compared with NC group,while the overexpression of GRP78 enhanced the proliferation of hepatoma cells.The same interference with the expression of SARA inhibited the proliferation of HCC cells,while the overexpression of SARA enhanced the proliferation of hepatoma cells.5.The results of recovery experiment showed that after interfering with GRP78,the expression of SARA decreased and the ability of cell proliferation decreased.However,overexpression of SARA while interfering with GRP78 could reverse the decrease of proliferation caused by interference with GRP78.After overexpression of GRP78,the expression of SARA increased,and the ability of cell proliferation increased.However,overexpression of GRP78 and interference with the expression of SARA can reduce the increase of proliferation caused by overexpression of GRP78.After interfering with GRP78,the protein level of Smad2/3 remained unchanged and the protein level of P-Smad2/3 decreased.On the contrary,the protein level of Smad2/3 remained unchanged and the protein level of P-Smad2/3 increased after overexpression of GRP78.Conclusion:GRP78 can affect the activation of TGF-βsignaling pathway in hepatocellular carcinoma.Further experiments found that GRP78 may promote the proliferation of hepatocellular carcinoma by up-regulating SARA,promoting the phosphorylation of Smad2/3 and activating TGF-βsignaling pathway. |
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