GRP78 Activates WNT/HOXB9 Pathway To Promote Invasion And Metastasis Of Hepatocellular Carcinoma By LRP6

Background:Hepatocellular Carcinoma(Hepatocellular Carcinoma,HCC)is one of the most common malignancies in the world and the third most likely cause of cancer-related death.Although some progress has been made in improving the prognosis of HCC patients,the 5-year postoperative survival rate is still low.The high mortality of HCC is mainly the result of intrahepatic and distal metastases.However,the mechanism of liver cancer invasion and metastasis is not clear.GRP78(Glucose-regulated Protein78,GRP78)is a type of stress Protein involved in the regulation of many important cellular functions.Such as Protein processing and folding,ER translocation,ER Associated Degradation(Endoplasmic Reticulum Associated Degradation,ERAD)and The Unfolded Protein Response(The Unfolded Protein Response,UPR)pathway.Changes in tumor microenvironment such as hypoxia and acidosis can induce high expression of GRP78,which helps cancer cells adapt to the new environment.Previous studies have shown that GRP78 plays a crucial role in the occurrence and development of liver cancer.As a sequence specific nuclear transcription factor,HOXB9(Homeobox B9,HOXB9)is involved in cell invasion,metastasis and differentiation.There is increasing evidence that the overexpression of HOXB9 is closely related to tumor growth and distant metastasis.For example,studies have found that HOXB9 is highly expressed in breast cancer and promotes the occurrence of breast cancer and lung metastasis.A recent study showed that HOXB9 was overexpressed in lung adenocarcinoma and predicted poor prognosis.In addition,our earlier studies revealed that HOXB9 was also over-expressed in liver cancer.The WNT signaling pathway is a highly conserved pathway in human embryonic development and tumorigenesis.This pathway also plays an important role in the invasion and metastasis of liver cancer.Recent studies have shown that HOXB9 is a target gene of WNT signaling pathway in breast cancer cells.Importantly,our previous studies have shown that the WNT/ HOXB9 signaling pathway can promote the invasion and metastasis of liver cancer.In addition,studies have shown that GRP78 is involved in the regulation of WNT signaling pathway.However,the specific molecular biological mechanism by which GRP78 regulates the WNT signaling pathway in liver cancer cells remains unclear.Objective:To explore the relationship between GRP78 and HOXB9 in regulating the invasion and metastasis of liver cancer cells and the specific molecular biological mechanism.Methods:1.106 cases of liver cancer tissues and corresponding paracancer tissue samples of patients who were admitted to the second affiliated hospital of nanchang university and diagnosed with hepatocellular carcinoma from February 2012 to February 2018 were collected.By real-time fluorescent Quantitative PCR(Quantitative Real Time Polymerase Chain Reaction,q RT-PCR),immunohistochemistry(IHC)Immunohistochemical,Western blot in the detection of liver cancer and corresponding adjacent tissues of GRP78 and HOXB9 m RNA and protein expression,correlation analysis are used to observe the correlation of GRP78 and HOXB9.The correlation between the expression of GRP78 and HOXB9 and the clinicopathological characteristics was analyzed,and kaplan-meier method was used to construct the patient survival curve.2.explore the expression of GRP78 and HOXB9 liver cancer cell height,selection of GRP78 protein expression is relatively high and the expression of relatively low each two cell lines,and then in the GRP78 high GRP78 expression of cell interference,low in the GRP78 expression of cell in the expression of GRP78,detection of GRP78 and HOXB9 m RNA and protein changes,and Transwell invasion and migration experiments to observe the changes of liver cancer cell invasion and metastasis ability.In vivo experiments,a stable cell line interfering GRP78 was constructed with lentivirus,and an orthotopic implantation model of liver cancer in nude mice was constructed with this cell line.The number of cancer nests of intrahepatic metastasis and lung metastasis in nude mice was observed by HE staining.3.The upstream and downstream relationship between GRP78 and HOXB9 was detected by in vitro and in vivo recovery experiments.In vitro experiments,in liver cancer cells with high GRP78 expression,interference with GRP78 also increased the expression of HOXB9.Correspondingly,in liver cancer cells with low GRP78 expression,overexpression of GRP78 also interfered with the expression of HOXB9,so as to observe the changes of m RNA and protein of GRP78 and HOXB9.Meanwhile,Transwell invasion and metastasis experiments were used to observe the changes of invasion and metastasis ability of liver cancer cells.In vivo experiments,HOXB9 was stably overexpressed in cell lines that were stable to interfere with GRP78,and an orthotopic implantation model of liver cancer in nude mice was established with this cell line.The number of cancer nests with intrahepatic metastasis and lung metastasis in nude mice was observed by HE staining.4.Explore the molecular biological mechanism of GRP78 regulating HOXB9.Bioinformatics analysis was used to find the possible pathway by which GRP78 regulates HOXB9,and the relevant results were verified.We confirmed that GRP78 regulates HOXB9 by activating the signal pathway through laser confocal,Western blot,immunoprecipition and other methods.Then,the specific molecular biological mechanism of GRP78 regulating signaling pathway in liver cancer cells was further explored.Results:1.Immunohistochemistry,Western blot and qrt-pcr results of liver cancer patients showed that GRP78 was highly expressed in 70.75%(75/106)of liver cancer patients,while HOXB9 was highly expressed in 53.77%(57/106)of liver cancer patients.Correlation analysis revealed that m RNA and protein expressions of GRP78 and HOXB9 were positively correlated and closely correlated with tumor diameter,tumor capsule,tumor microsatellite focus and venous invasion of liver cancer patients(P<0.05).Survival curves of patients showed that postoperative survival rate of liver cancer patients with high expression of GRP78 and HOXB9 was lower(P<0.05).2.Cell screening results showed that m RNA and protein expression levels of GRP78 and HOXB9 in five liver cancer cells(Huh-7,SMCC7721,MHCC97 H,Hep3B,HCCLM3)were significantly higher than that of normal liver cell line hl-7702.Interference with GRP78 expression in HCCLM3 and MHCC97 H cells showed that m RNA and protein levels of GRP78 and HOXB9 were decreased simultaneously(P<0.05).Transwell test results showed that interference with GRP78 reduced the invasion and metastasis of HCC cells.In the orthotopic implantation model of liver cancer in nude mice,the number of nests with intrahepatic metastasis and lung metastasis in nude mice implanted with stable interfering GRP78 cell lines was lower than that in the control group implanted with normal cell lines(P<0.05).3.The response experiment results showed that after GRP78 interference,the expression of HOXB9 decreased,and the ability of cell invasion and metastasis decreased.However,when interfering with GRP78,overexpression of HOXB9 can restore the reduced invasion and metastasis ability caused by interfering GRP78.After overexpression of GRP78,the expression of HOXB9 increased,and the ability of cell invasion and metastasis increased.However,interfering HOXB9 expression with overexpression of GRP78 can reduce the increase of invasion and metastasis ability caused by overexpression of GRP78.The orthotopic implantation model of liver cancer in nude mice showed that in the experimental group with stable GRP78 interference and overexpression of HOXB9,the number of tumors with liver metastasis and lung metastasis in nude mice was higher than that in the control group with only stable GRP78 interference.4.Bioinformatics IPA pathway analysis results showed that GRP78 may regulate the expression of HOXB9 through the WNT/ beta-catenin signal pathway,and further experiments found that interfering GRP78 could reduce the expression of beta-catenin and reduce the entry of-catenin into the nucleus,thereby inhibiting the WNT/ beta-catenin signal pathway.5.Further molecular biological mechanism studies found that GRP78 can promote the maturation of LRP6 and protect LRP6 from degradation by the ERAD pathway,thereby promoting the expression of LRP6 and activating the WNT/beta-catenin signal pathway.Conclusion:GRP78 can promote the maturation and membrane expression of LRP6 and protect LRP6 from ERAD degradation by acting as a molecular chaperone of LRP6,thereby activating the WNT/ beta-catenin signal pathway to promote the expression of HOXB9 and enhance the invasion and metastasis of hepatocellular carcinoma.This molecular mechanism provides a new insight into the mechanism of invasion and metastasis of liver cancer cells and a new basis for molecular targeted therapy of liver cancer.