The Effect And Molecular Mechanism Of CREB3L4 In Hepatocellular Carcinoma

Research BackgroundPrimary liver cancer is one of the common malignant tumors worldwide and is the third most common cause of death from cancer in the population,accounting for 8.3%of all cancer deaths in 2020.Hepatocellular liver cancer has an insidious onset,the symptoms are not obvious,and most patients are already at an advanced stage once they are detected,losing the valuable opportunity of surgical treatment and unable to undergo radical resection.The high malignancy,early metastasis,easy recurrence and insensitivity to radiotherapy of hepatocellular liver cancer also seriously threaten the life safety of patients,therefore,it is urgent to investigate the mechanism of HCC development and the development of new drug targets for HCC treatment.Sorafenib,a small molecule multikinase inhibitor,is currently the only approved drug for the treatment of patients with advanced hepatocellular liver cancer.It inhibits tumor cell proliferation and angiogenesis and promotes tumor cell apoptosis by inhibiting the activity of Raf-1 and VEGFR and interfering with cellular signaling pathways mediated by the Raf-1 and VEGFR pathways.Although sorafenib can prolong the median survival time of patients with advanced HCC,this benefit is reduced by the development of tumor cell resistance to sorafenib,which ultimately leads to treatment failure.The development of sorafenib resistance has become a major obstacle in the clinical treatment of patients with advanced HCC at this stage,and it is therefore particularly important to fully understand the mechanisms by which resistance to sorafenib therapy develops in HCC patients.The CREB3 transcription factor family consists of five members in mammals:CREB3(LZIP or Luman),CREB3L1(OASIS),CREB3L2(BBF2H7),CREB3L3(CREB-H)and CREB3L4(AIbZIP,TISP40),and they also belong to the basic leucine zipper transcription The CREB3 subfamily transcription factors have typical structural features that regulate RIP activation.The CREB3L4 is a member of the CREB3 family and was originally discovered by Qi et al.in 2002.It is encoded by the CREB3L4 gene located on human chromosome 1q21.3,whose structural domains from N-terminal to C-terminal include:a trans-activation domain mediating sequence-specific DNA binding,a conserved domain ATB consisting of about 30 residues(adjacent to bZIP),a basic region next to the ZIP structural domain,the bZIP DNAbinding domain,and a transmembrane structure.Similar to other CREB3 family members,CREB3L4 regulates the expression of target genes through its cAMP response element and thus participates in complex physiopathological processes such as metabolism,development,and tumor formation.Studies have shown that CREB3L4 has an important regulatory role in tumorigenesis and development.For example,CREB3L4 is involved in regulating the progression of prostate cancer promoted by androgen receptor and inositol-dependent kinase 1 α.It has also been found that CREB3L4 promotes gastric cancer progression and angiogenesis through transcriptional activation of the VEGFA promoter.The above studies show that CREB3L4 is overexpressed in prostate,breast and gastric cancers,and its aberrant activation can significantly promote malignant progression of tumors,suggesting that CREB3L4 is involved in regulating disease progression of malignant tumors.In addition,it has been shown that the mRNA level of CREB3L4 was significantly elevated in hepatocellular carcinoma tissues compared with normal liver tissues,but its regulatory mechanism on the development of hepatocellular carcinoma and the development of resistance to sorafenib treatment has not been reported yet.Purpose of the study(1)To explore the expression level of CREB3L4 in HCC cancer tissues.(2)To explore the role and molecular mechanism of CREB3L4 in the progression of HCC.(3)To explore the molecular mechanism of CREB3L4 on the development of resistance to sorafenib for HCC treatment.Part Ⅰ Expression of CREB3L4 in hepatocellular liver cancer and its impact on biological functionsMaterial Method1.Database prediction of CREB3L4 expression levels in HCC tissues and the relationship with prognosisThe GEPIA database is a commonly used tumor search database to query the expression of multiple genes in different tumors and the correlation with different factors.Therefore,the prediction of CREB3L4 expression in HCC tissues and the relationship between CREB3L4 and prognosis of HCC patients were performed in the database.2.Detection of CREB3L4 expression levels in HCC tissues and the relationship withpatient prognosisParaffin sections of 83 hepatocellular carcinomas and their corresponding distal noncancerous liver tissues were applied for immunohistochemical staining of CREB3L4 molecules to detect their expression localization and expression levels in hepatocellular carcinoma cells.After staining,Image J software was applied to further analyze the expression differences of CREB3L4 in hepatocellular carcinoma tissues and their paired non-cancerous liver tissues.The relationship with the clinicopathological stage of patients was also analyzed according to the expression differences.The western blot method was applied to detect the expression levels of CREB3L4 protein in hepatocellular carcinoma tissues and their paired non-cancerous liver tissues of 20 patients with hepatocellular carcinoma.Image J software was applied to quantify CREB3L4 expression bands in liver cancer tissues and their corresponding non-cancerous liver tissues,and GraphPad Prism 9.3 software was applied to statistically analyze the grayscale of the bands.3.Detection of the regulatory effect of CREB3L4 on the proliferation of HCC cellsHepatocellular carcinoma cell lines HUH7 and Hep3B were transfected with specific CREB3L4 short hairpin RNA(shCREB3L4),respectively,and non-specific nonsense sequence(shNC)transfected groups were used as controls.western blot,qRT-PCR to identify the interference efficiency were used to construct CREB3L4 expression suppressed cell models.Cancer cell lines HUH7 and Hep3B were transfected with HA-CREB3L4 plasmids,respectively.western blot,qRT-PCR to identify transfection efficiency were used to construct CREB3L4 overexpression cell models.CCK-8 assay and EdU were used to detect the changes of cell proliferation ability in CREB3L4 expression suppressed cell model and overexpression cell model,and further detect the changes of cell colony formation ability in CREB3L4 expression suppressed cell model and overexpression cell model.4.Detection of CREB3L4 as a regulator of HCC tumor stem cell markersqRT-PCR was performed to detect the mRNA expression levels of CREB3L4 expression in HUH7 and Hep3B cell lines in suppressed cell models and cells in overexpression cell models □CD24,CD44,CD47,CD133 and AFP.5.Construction of a nude mouse xenograft tumor model to detect the regulatory effect of CREB3L4 on HCC0.1 ml of 10^7 HUH7 cells were injected subcutaneously into the unilateral groin of nude mice.When tumors were visible to the naked eye,the shCREB3L4 plasmid was injected into the tumors.Mice were executed at day 28 after the first injection and tumors were stripped from the mice for further analysis.Tumor tissues were extracted for tissue sectioning,and immunohistochemical staining was applied to detect successful interference with CREB3L4 and KI67 expression in the tumor tissue.The tumor weights were weighed and the tumor volumes were measured to compare and analyze the differences in tumor weights and volumes between the experimental and control groups.Experimental results1.Database predicts that CREB3L4 is highly expressed in HCC tissues and correlates with poor prognosisA search of the GEPIA database revealed that CREB3L4 expression in HCC tissues was higher than that in normal liver tissues,and in terms of five-year overall survival and diseasefree survival,patients with high CREB3L4 expression appeared to have slightly lower survival than those with low CREB3L4 expression,although there was no statistical difference,suggesting that perhaps CREB3L4 expression is associated with HCC patient poor prognosis was associated.2.High expression of CREB3L4 in HCC tissues and correlation with clinicopathological factorsThe results of immunohistochemical staining experiments revealed that CREB3L4 was not expressed or showed low expression in normal liver tissues,while it was highly expressed in HCC tissues and its expression was located in the nucleus.Based on the microarray immunohistochemical staining results,we performed a statistical analysis and found that the expression of CREB3L4 was positively correlated with the TNM stage of HCC patients(P<0.05).Similarly,the results of immunohistochemical staining experiments of 83 HCC patients were grouped by median CREB3L4 expression and divided into CREB3L4 high expression group and low expression group.Survival curves were made between the two groups,and the analysis revealed that the survival of patients in the high expression group was significantly lower than that of patients in the low expression group,suggesting that high CREB3L4 expression was positively correlated with tumor stage and poor prognosis of HCC.Protein immunoblotting assay was performed on liver cancer tissues and normal tissues adjacent to cancer in 20 pairs of HCC patients.The results revealed that the expression of CREB3L4 was significantly higher in HCC tissues compared to paraneoplastic tissues.3.CREB3L4 promotes the proliferative capacity of HCC cellsAfter successfully establishing the cell model of CREB3L4 overexpression and interference,we firstly examined the effect of CREB3L4 on the proliferative activity of HCC cells using CCK-8 proliferation assay.The results showed that overexpression of CREB3L4 in HUH7 and Hep3B cells increased the proliferative activity of the cells;interference with CREB3L4 expression in HUH7 and Hep3B cells decreased the proliferative activity of the cells.To further verify the effect of CREB3L4 on cell proliferation ability,we further refined the colony formation assay.It was found that the number of colony formation and the volume size of colonies in HUH7 and Hep3B cells after up-regulation of CREB3L4 expression were higher than those in the null control group;the number of colony formation and the volume size of colonies in HUH7 and Hep3B cells after down-regulation of CREB3L4 expression were lower than those in the negative control group.Finally,we performed another EdU assay to verify the effect of CREB3L4 on cell proliferation ability.The results showed that the cells of HUH7 and Hep3B cells in the proliferative division phase after up-regulation of CREB3L4 expression were significantly higher than those of the null control;the cells of HUH7 and Hep3B cells in the proliferative division phase after down-regulation of CREB3L4 expression were significantly lower than those of the negative control.In summary,the results of CCK-8 proliferation assay,colony formation assay and EdU assay were consistent and together confirmed that CREB3L4 could promote the proliferative ability of HCC cells.4.CREB3L4 increases the level of HCC tumor stem cell markers and promotes tumor malignancyThe qRT-PCR assay was performed on liver cancer cancer stem cell markers,and the results showed that the levels of CD24,CD44,CD47,CD 133 and AFP were significantly higher in HUH7 cells after up-regulation of CREB3L4 expression than in the null controls;CD44,CD 133 and AFP were significantly lower in HUH7 cells after down-regulation of CREB3L4 expression than in the negative controls.5.CREB3L4 significantly promotes the growth of xenograft tumors in nude miceAfter successful construction of the xenograft model in nude mice,shCREB3L4 plasmid was injected intratumorally,and the shNC plasmid injection group was used as the control.Nude mice were executed 28 days after plasmid injection,and tumors were isolated,measured and weighed in volume.Subsequent immunohistochemical staining verified the successful interference of CREB3L4 in the shCREB3L4 transfected group.The results of tumor analysis showed that the tumor size and volume in the shCREB3L4-injected group were significantly smaller than those in the shNC-injected group.In addition the tumor mass of shCREB3L4 transfected group was much smaller than that of shNC transfected group.Meanwhile,inhibition of CREB3L4 expression could reduce Ki67 expression.The above results suggest that CREB3L4 significantly promotes the growth of xenograft tumors in nude mice.Part Ⅱ CREB3L4 promotes the biological function of hepatocellular liver cancer through mTORCl signaling pathwayMaterial Method1.Detection of signal transduction pathways in which CREB3L4 exerts its effect in HCCAfter overexpression or inhibition of CREB3L4 expression,western blot was applied to detect changes in signaling pathways including mTROC1,MAPK,AKT,NF-κB,etc.The successfully constructed CREB3L4 overexpression cell model was treated with mTORC1 pathway inhibitor(Rapamycin,100 nM)and western blot was applied to detect the changes of mTOR signaling pathway related proteins.2.Investigating the specific mechanism of CREB3L4 regulation in the proliferative capacity of HCCCellular functional recovery assay was performed by transfecting HUH7 and Hep3B cells with HA-CREB3L4 plasmid to construct CREB3L4 overexpression cell model and stimulated with mTORC1 specific blocker Rapamycin.CCK-8 assay was performed to detect the change of cell proliferation ability after stimulation with Rapamycin in CREB3L4 overexpression cell model.The changes in cell colony formation ability of CREB3L4 overexpression cell model stimulated by Rapamycin were further examined.3.Probing the specific mechanism of CREB3L4 activation of mTORC1 signaling pathway 3.1 Probing the correlation between CREB3L4 and RhebThe GEPIA database is a commonly used tumor search database to query the expressionof multiple genes in different tumors and their correlation with specific molecules.Therefore,the prediction of Rheb expression in HCC tissues and the relationship between Rheb and CREB3L4 was performed in the database.Subsequently,to investigate whether CREB3L4 regulates the transcription of Rheb and thus the mTORC1 signaling pathway,we transfected HA-CREB3L4 plasmids in HUH7 and Hep3B cells to construct a cellular model of CREB3L4 overexpression and detected the expression of Rheb by qRT-PCR and western blot.To elucidate the regulatory effect of CREB3L4 on Rheb,we performed in vivo experiments to detect the effect of CREB3L4 on a xenograft tumor model in nude mice by subcutaneous injection of HUH7 cells into the unilateral groin of the nude mice.When tumors were visible to the naked eye,20ug shCREB3L4 plasmid was injected into the tumors on the left side every other day,and 20ug shNC plasmid was injected into the tumors in the control group every other day until the mice were executed on day 28 after the first injection.western blot was performed to detect the expression of Rheb,p-mTORC1,mTORC1,and CREB3L4 molecules in the tumor tissues.To further validate the expression of Rheb and p-mTORC1 in clinical samples,we examined 20 clinical HCC samples and paired paracancerous tissue samples by western blot.3.2 Probing the specific mechanism of CREB3L4 activation of mTORCl signaling pathwayThe JASPAR database was analyzed for the predicted binding site of CREB3L4 to Rheb,and ChIP-PCR primers were designed and ChIP-PCR experiments were performed for the predicted binding site of transcription factor CREB3L4.Subsequently,a dual luciferase reporter gene assay was performed to detect the binding ability of CREB3L4 bound to the promoter region of Rheb gene.Experimental results1.CREB3L4 positively regulates the mTORCl signaling pathwayAfter successfully establishing the cell model of CREB3L4 overexpression and interference,the results of Western blot experiments suggested that the phosphorylation level of mTORC1 increased significantly after upregulating CREB3L4 expression,and its downstream S6K1,S6,4E-BP1 phosphorylation level also increased significantly;after interfering with CREB3L4 expression,the phosphorylation level of mTORC1 decreased significantly,and its downstream S6K1,S6,and 4E-BP1 phosphorylation levels also decreased significantly.To further confirm that CREB3L4 has a regulatory effect on the mTOR signaling pathway,an inhibitor of the mTORC1 pathway was added to the cell model of CREB3L4 overexpression and interference Rapamycin.Subsequently,it was found by Western blot assay that the specific inhibitor of the mTORC1 signaling pathway could reverse the activation of the mTOR signaling pathway by CREB3L4.The above results suggest that CREB3L4 positively regulates the mTORC1 signaling pathway in HCC.2.CREB3L4 promotes the proliferative capacity of HCC cells through the mTORC1 signaling pathwayAfter successfully establishing a cell model of CREB3L4 overexpression and interference,Rapamycin,an inhibitor of the mTORC1 pathway,was added for stimulation.Firstly,we tested whether the regulation of CREB3L4 on the proliferative activity of HCC cells through the mTORC1 signaling pathway using CCK-8 proliferation assay.It was found that overexpression of CREB3L4 in HUH7 and Hep3B cells increased the proliferative activity of the cells,but administration of Rapamycin stimulation reversed the increase in proliferative activity by CREB3L4.To further verify the effect of CREB3L4 on cell proliferation capacity,we further refined the colony formation assay.It was found that the number of colony formation and the volume size of colonies were higher in HUH7 and Hep3B cells after upregulation of CREB3L4 expression than in the no-load control group,but Rapamycin stimulation reversed the changes in the number of colony formation and the volume size of colonies brought about by CREB3L4.In summary,the results of CCK-8 proliferation assay and colony formation assay were consistent and together confirmed that CREB3L4 promotes the proliferative ability of HCC cells through the mTORC1 signaling pathway.3.CREB3L4 activates the mTORCl signaling pathway by regulating the transcription of Rheb3.1 CREB3L4 was positively correlated with Rheb expression in HCCWe found that Rheb expression levels were high in HCC tissues in 369 cancer patients using 50 normal tissues as control by online data analysis.We also analyzed these cases by staging and found that Rheb expression levels increased with increasing tumor stage but decreased at stage IV in HCC tissue samples.And the correlation analysis of CREB3L4 with Rheb revealed that the two molecules were positively correlated in HCC tissues.After successfully establishing the cell model of CREB3L4 overexpression and interference,the expression of Rheb was detected by qRT-PCR and western blot.The results showed that Rheb expression increased after overexpression of CREB3L4.Conversely,Rheb expression was decreased after inhibition of CREB3L4.After successful construction of the xenograft model in nude mice,shCREB3L4 plasmid was injected intratumorally,and the shNC plasmid injection group was used as the control.After 28 days of plasmid injection,the nude mice were executed,tumors were isolated,and tumor protein tissues were extracted.western blot was performed to detect the expression of Rheb,p-mTORC1,mTORC1,CREB3L4 molecules in tumor tissues.The results showed that the expression of Rheb and p-mTORC1 molecules were reduced in shCREB3L4 group.To further verify the expression of Rheb and p-mTORC1 in clinical samples,we examined 20 clinical HCC samples and paired paraneoplastic tissue samples by western blot.The results showed that both Rheb and p-mTORC1 expression were upregulated in HCC tissues compared to normal tissues adjacent to cancer.Together,the above experiments indicate that CREB3L4 is positively correlated with Rheb expression.3.2 CREB3L4 regulates the transcriptional level of Rheb by binding to the promoter of Rheb geneChIP-PCR primers were designed and ChIP-PCR experiments were performed for the predicted binding site of transcription factor CREB3L4.The results showed that CREB3L4 was significantly enriched at the predicted promoter region position of Rheb gene in HUH7 cells.We then performed dual luciferase reporter gene experiments,which showed that CREB3L4 promotes Rheb transcription by binding to the promoter region of the Rheb gene in HUH7 cells.Part Ⅲ CREB3L4 reduces the chemosensitivity of sorafenib for HCC by regulating the mTORCl signaling pathwayMaterial Method1.Investigating whether CREB3L4 is involved in regulating the sensitivity of HCC cells to Sorafenib chemotherapy1.1 Probing the role of overexpression of CREB3L4 on the sensitivity of Sorafenib chemotherapyCell models of CREB3L4 overexpression were constructed by transfecting HA-CREB3L4 plasmid in HUH7 and Hep3B cells,and cell activity was assayed after stimulation with Sorafenib.CCK-8 assay was used to detect changes in cell activity of CREB3L4 overexpression cell models after stimulation with Sorafenib,and further to detect changes in CREB3L4 overexpression cell models after stimulation with Sorafenib.The CCK-8 assay was used to detect changes in the cellular activity of the CREB3L4 overexpression cell model after stimulation with Sorafenib,and further to detect changes in the colony forming ability of the CREB3L4 overexpression cell model after stimulation with Sorafenib.1.2 Probing the role of CREB3L4 inhibition on Sorafenib chemosensitivityCell models of CREB3L4 interference were constructed by transfecting shCREB3L4 plasmid in HUH7 and Hep3B cells,and cell activity was assayed after stimulation with Sorafenib.CCK-8 assay was performed to detect changes in cell activity of CREB3L4 interference cell models stimulated by Sorafenib,and further to detect changes in CREB3L4 interference cell.The CCK-8 assay was used to detect changes in the cellular colony-forming ability of the CREB3L4-interfering cell model after stimulation with Sorafenib.2.Investigating the specific mechanism by which CREB3L4 regulates the sensitivity of HCC cells to Sorafenib chemotherapyCell models of CREB3L4 overexpression or interference were constructed in HUH7 and Hep3B cells transfected with HA-CREB3L4 or shCREB3L4 plasmids,and protein extraction was performed after stimulation with Sorafenib.western blot was performed to detect CREB3L4,Rheb,mTORC1,S6K1,S6,4E-BP1 and other molecules were expressed in HCC cells.3.Nude mouse xenograft tumor model to detect the effect of CREB3L4 on the sensitivity of Sorafenib chemotherapyKnock-down stable transgenic cell lines of the HUH7 cell line were first established using a viral volume of viral MOI=10.The HUH7 knockdown-low stable-transformation cell line was injected subcutaneously through the unilateral groin of nude mice.The mice were continuously observed daily and Sorafenib(5 mg/kg)was injected into the tumor on the left side every 4 days when a tumor was visible to the naked eye and an equal volume of PBS was injected into the tumor in the control group every 4 days until the mice were executed on day 28 after the first injection.Tumors were isolated from the groin of the mice for further analysis.The weight of the peeled tumor tissue was weighed and the tumor volume was measured to compare and analyze the difference in tumor weight and volume between the experimental and control groups.Experimental results1.CREB3L4 attenuates the sensitivity of HCC cells to Sorafenib chemotherapy1.1 Overexpression of CREB3L4 significantly inhibited the sensitivity of HCC cells to Sorafenib chemotherapyAfter successfully establishing a cell model of CREB3L4 overexpression and interference,Rapamycin,an inhibitor of the mTORC1 pathway,was added for stimulation.Firstly,we examined the effect of CREB3L4 on the effect of conventional chemotherapeutic drug Sorafenib in hepatocellular liver cancer using CCK-8 proliferation assay.The results showed that with the increasing concentration of Sorafenib stimulation,the cell activity decreased in both HA-CREB3L4 and control groups,but the cell activity in the control group was lower than that in HA-CREB3L4 group.When Sorafenib(10 μM)was given for 48 h of treatment,the results suggested that the cell proliferation activity was diminished after the addition of Sorafenib to Mock-Sorafenib compared with the Mock-DMSO group;the cell activity was enhanced in the CREB3L4-DMSO group compared with the Mock-DMSO group;and compared with Mock-Sorafenib,cell activity was enhanced in the CREB3L4-Sorafenib group with sorafenib administration.We subsequently performed colony formation assay,and found that the number of cell colonies formed and the volume size of the colonies decreased after the addition of sorafenib in the Mock-sorafenib compared to the Mock-DMSO group,and the number of cell colonies formed and the volume size of the colonies increased in the CREB3L4-DMSO group compared to the Mock-DMSO group.When sorafenib was given to the CREB3L4-sorafenib group compared with Mock-sorafenib,the number of cell colony formation and the volume size of the colony increased increased.Taken together,the above experiments showed that overexpression of CREB3L4 significantly inhibited the chemosensitivity of sorafenib to HUH7 and Hep3B cells.1.2 Inhibition of CREB3L4 significantly reduces the sensitivity of HCC cells to Sorafenib chemotherapyAfter successful establishment of a CREB3L4-disrupted cell model,Rapamycin,an inhibitor of the mTORC1 pathway,was added for stimulation.Firstly,we examined the effect of CREB3L4 on the effect of Sorafenib,a conventional chemotherapeutic agent for hepatocellular liver cancer,using CCK-8 proliferation assay.The results showed that with increasing concentration of Sorafenib stimulation,the cell activity decreased in both shCREB3L4 and control groups,but the cell activity in the control group was higher than that in the shCREB3L4 group.When Sorafenib(10μM)was given for 48 h of treatment,the results suggested that the cell proliferation activity was diminished in the shNC-Sorafenib addition compared to the shNC-DMSO group;the cell activity was diminished in the shCREB3L4DMSO group compared to the shNC-DMSO group;the cell activity was diminished in the shCREB3L4-DMSO group compared to the shNC-Sorafenib compared to shNC-Sorafenib,and diminished cellular activity in the shCREB3L4-Sorafenib group given Sorafenib.We subsequently performed colony formation experiments and found that the number of cytosolic colony formation and the volume size of the colony decreased when shNC-Sorafenib was added to Sorafenib compared to the shNC-DMSO group,and the number of cytosolic colony formation and the volume size of the colony decreased when shCREB3L4-DMSO was added to shNC-Sorafenib compared to the shNC-DMSO group.When Sorafenib was given to the CREB3L4-Sorafenib group compared with shNC-Sorafenib,the number of cytosolic colony formation and the volume size of the colony were reduced.Taken together,the above experiments showed that inhibition of CREB3L4 significantly enhanced the chemosensitivity of Sorafenib on HUH7 and Hep3B cells.2.CREB3L4 regulates the sensitivity of HCC cells to Sorafenib chemotherapy through activation of the Rheb/mTORCl signaling pathwayTo further clarify the molecular mechanism of CREB3L4 regulation of chemo-sensitivity in HCC cells,we performed western blot assays.The expression of CREB3L4 was up-regulated by transfection of HA-CREB3L4 plasmid in HUH7 and Hep3B cell lines and treated with Sorafenib.The results showed that the CREB3L4-Sorafenib group given Sorafenib upregulated the Rheb/mTORC1 signaling pathway compared to Mock-Sorafenib.We subsequently transfected shCREB3L4 plasmids in HUH7 and Hep3B cell lines to interfere with CREB3L4 expression and treated with Sorafenib.The results showed that the Rheb/mTORC1 signaling pathway was downregulated in the shCREB3L4-Sorafenib group given Sorafenib compared to shNC-Sorafenib.Taken together,CREB3L4 attenuates the sensitivity of HCC cells to Sorafenib chemotherapy by activating the Rheb/mTORC1 signaling pathway3.CREB3L4 significantly attenuates the inhibitory effect of Sorafenib on the growth of xenograft tumors in nude miceWe used viral MOI=10 or 20 in viral volume to establish knock-down stable transfection cell lines of HUH7 cell line.Cell fluorescence intensity was observed after 48h of cell infection conditions to determine transfection efficiency.When cultured to 72h,stimulation was performed using a concentration of 5ug/ml puromycin,and puromycin was replaced at 2-day intervals to obtain stable-earned cell lines for 2 weeks of continuous stimulation.Subsequently,a nude mouse xenograft model was constructed using the HUH7 knockdown low-stable cell line followed by injection of Sorafenib(5 mg/kg)into the tumors on the left side every 4 days and an equal volume of PBS into the control tumors every 4 days until the mice were executed on day 28 after the first injection.Tumors were isolated from the groin of the mice for further analysis.The results of tumor analysis showed that the tumor size and volume in the shCREB3L4+Sorafenib group were significantly smaller than those in the shNC+Sorafenib group.In addition,the tumor mass in the shCREB3L4 transfected group was much smaller than that in the shNC transfected group.Conclusion1.CREB3L4 is highly expressed in HCC patients and is associated with TNM stage and poor prognosis of the tumor2.CREB3L4 promotes the proliferative capacity of HCC and tumor development3.CREB3L4 promotes the proliferative capacity of HCC cells by regulating the transcription of Rheb,which in turn activates the mTORC1 signaling pathway4.CREB3L4 significantly inhibits the sensitivity of HCC cells to Sorafenib chemotherapy5.CREB3L4 regulates the sensitivity of HCC cells to Sorafenib chemotherapy through activation of Rheb/mTORC1 signaling pathwayInnovativeness and significance1.This project is the first to report that the transcription factor CREB3L4 tumor-promoting factor exerts a pro-cancer effect in HCC progression,providing a new molecular biological marker and therapeutic target for the clinical diagnosis and treatment of HCC.2.This study is the first to report that CREB3L4 promotes the proliferation of HCC cells by controlling the transcription of Rheb,which in turn activates the mTORC1 signaling pathway and exerts a pro-cancer effect on HCC,laying the foundation for elucidating the mechanism of hepatocellular carcinoma progression and effectively regulating it at the molecular level.3.This topic reports for the first time that CREB3L4 acts as a chemotherapeutic modulator toattenuate the sensitivity of HCC cells to Sorafenib,laying the foundation for enhanced treatment response and improved prognosis in clinical hepatocellular carcinoma patients.