| BackgroundPrimary liver cancer is a highly malignant tumor.Hepatocellular carcinoma is the main type and has become one of the most common malignant tumors in the world.According to the latest statistics,hepatocellular carcinoma is the fifth common malignant tumors and the third leading cause of cancer death,with about 600,000 new cases annually worldwide.Hepatocellular carcinoma 1s characterized by late detection,rapid progress,and poor prognosis.At present,more than half of the cases of hepatocellular carcinoma in the world occur in China,which seriously threatens the health of Chinese.The occurrence and development of hepatocellular carcinoma is a long-term,complex and continuous process caused by many factors.The etiological factors include alcohol,hepatitis virus infection and so forth,which ultimately lead to tumorigenesis via a variety of complex gene mutations.Therefore,to explore complex pathogenesis,it is crucial to seek a valuable gene in hepatocellular carcinoma.Synaptptagmin(Syt)1s a type of secreted vesicle-like protein,which consists of a N-terlinal transmembrane domain and a C-terninal C2 domain that binds to Ca2'(including CZA and C2B).The Ca2+-bound synaptic binding protein subtype regulates the expansion of fusion pores of neurons,neuroendocrine cells,and various other secretory cells.Synaptotagmin 7(Syt7)is a member of the family and few studies reported the role of Syt7 in tunors.It has been reported that Syts are abnormally expressed in several types of tumors.For example,Syt(not mentioned specific Syt molecule)is elevated in small cell lung cancer,and the expression of Sytl3 in the left colon cancer sample is lower than the right.Sytl is highly expressed in neuroblastoma cells,and the inhibitory effect of calmodulin on parathyroid hormone contributes to the loss of expression of Sytl in parathyroid adenoma.Phosphorylation of extended Sytl(E-Sytl)is involved in the invasion pathway of proto-oncogene lung cancer fusion kinase activation.However,no studies have reported the role of Syt,especially Syt7,in hepatocellular carcinoma.Objective(1)To detect the protein expression of Syt7 in tumorous and adjacent non-tumorous tissues of hepatocellular carcinoma,and to analyze the correlation between the protein levels of Syt7 and clinicopathological parameters in hepatocellular carcinoma(2)To detect the mRNA level of Syt7 in hepatoma carcinoma cells.Methods(1)The paraffin-embedded specimens of hepatocellular carcinoma patients who were treated with surgical resection from September 2013 to February 2018 in the First Affiliated Hospital of Bengbu Medical College were collected.Immunohistochemistry was used to detect the protein expression of Syt7 in tumorous and adjacent non-tumorous tissues;(2)The expression level of Syt7 mRNA was detected by qRT-PCR;(3)The correlation between the protein expression level of Syt7 and clinical pathological parameters of hepatocellular carcinoma was retrospectively analyzed;(4)The comparison of the positive rate of Syt7 protein between tumorous and adjacent non-tumorous tissues and the correlation between Syt7 protein positive rate and clinicopathological parameters were analyzed by Pearson's chi-square test.The Kaplan-Meier model with log-rank test and Cox regression model were used to assess the factors associated with overall survival(OS)and disease-free survival(DFS).The difference was considered statistically significant provided P less than 0.05.Results(1)The expression level of Syt-7 was significantly higher in txumorous tissues(64.00%,48/75)than that in adjacent non-tumorous tissues(2.67%,2/75),and the difference was statistically significant(P<0.05);(2)The level of Syt-7 was significantly associated with AFP level(P=0.001),tumor size(P<0.001),tumor number(P?0.022),vascular invasion(P?0.008),TNM stage(P?0.003),and tumor differentiation(P<0.001);(3)Cox univariate analysis showed that tumor size,vascular invasion,TNM stage,tumor differentiation,Child-Pugh grade and expression of Syt7 were the main prognostic factors tor OS in patients with hepatocellular carcinoma.Multivariate analysis further showed that tumor diameter>5 cm(HR?4.22,95%Cl:1.44-12.39,P?0.009),vascular invasion(HR?2.71,95%Cl:1.15-6.40,P=0.023),Child-Pugh B grade(HR?4.16,95%Cl:1.54-11.25,P?0.005)and positive Syt7 expression(HR=2.84,95%Cl:1.08-7.49,P=0.035)were independent prognostic factors for OS;(4)Cox univariate analysis showed that AFP levels,tumor size,vascular invasion,TNM stage,tumor differentiation,Child-Pugh grade,and expression of Syt7 were the main prognostic factors for DFS.Multivariate analysis further showed that preoperative AFP? 200 ng/ml(HR=2.37,95%Cl:1.13-4.97,P=0.022),tumor diameter>5cm(HR?4.30,95%Cl:1.43-12.95,P=0.010),vascular invasion(HR?2.57,95%Cl:1.10-6.01,P-0.030),Child-Pugh B grade(HR?4.65,95%Cl:1.72-12.56,P=0.002)and positive Syt7 expression(HR?3.43,95%CI:1.26-9.29,P?O.016)were independent prognostic factors for DFS;(5)qRT-PCR showed that the mRNA level of Syt7 was highly expressed in Hep-7 and Hep-3B cells,and relatively lowly expressed in SMMC-7721,Hep G2 and BEL-7402 cells.Conclusions(1)The positive expression rate of Syt7 protein in tumorous tissues was significantly higher than that in adjacent tissues.The expression level of Syt7 was closely related to AFP level,tumor diameter,tumor number,vascular invasion,TNM stage and tumor differentiation degree in patients with hepatocellular carcinoma;(2)The expression of Syt-7 was an independent prognostic factor for OS and DFS in hepatocellular carcinoma;(3)The mRNA level of Syt7 was highly expressed in hepatoma cells Huh-7 and Hep-3B,and abundantly expressed in hepatoma cells SMMC-7721,Hep G2 and BEL-7402.BackgroundHepatocellular carcinoma is a common malignant tumor of the digestive system.The main etiology is drinking in western countries and hepatitis B virus infection in China.Because of its insidious onset,early symptoms are not obvious and early detection is difficult,which leads to low rate of radical surgical resection.Therefore,the comprehensive treatment is particularly important,including chemotherapy,gene-targeted therapy,and so on.Hepatocellular carcinoma is highly malignant with a poor prognosis.Currently,there is no satisfactory treatment.Researchers have shown that the occurrence and development of hepatocellular carcinoma is a complex process with multiple genes and multiple steps.In recent years,with the continuous advancement of molecular biology techniques,such as gene intervention technology,RNA interference technology,and so forth,it is possible to treat hepatocellular carcinoma by targeting gene.Therefore,it is crucial to study the target genes that play a key role in the process of proliferation,invasion and metastasis of hepatocellular carcinoma.We can further enrich the comprehensive treatment of hepatocellular carcinoma by specifically cutting or even replacing pathogenic genes or repairing abnormal genes.Some genes may be highly expressed in hepatocellular carcinoma cells,while it is key to explore whether they affect proliferation,cloning,cycle change,apoptosis,invasion,metastasis,etc.The exocytosis involved in the release of neurotransmitters from the nervous system and the release of insulin from islet beta cells is an important component of the organism's involvement in life activities and biochemical reactions.Various proteins play important roles in these physiological and biochemical processes.Protein transport between compartments within the cell is mediated by one organelle transporter and selectively fused to another organelle.The organelle-specific transport of vesicles requires proteins that regulate vesicle transport,localization,and fusion.According to the SNARE(soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor)hypothesis:each type of vesicle contains a v-SNARE(vesicle SNARE)and can only be associated with a t-SNARE(target SNARE)to a suitable receptor.Cell membrane.Among them,synaptoPhysin and VAMP(vesicle-associated membrane protein)belong to v-SNARE;while synaptic fusion protein and SNAP-25 family belong to t-SNARE.The SNARE protein is involved in all steps of the secretory pathway,but each SNARE protein has compartmental specificity and contributes to the specificity of the site and fusion.As a Ca2+high-affinity sensor,Syt7 is involved in non-neuronal cells including insulin-secreting granules of pancreatic p-cells,large,dense-centered vesicles,lysosomes,large vesicles and the regulation of fusion pore dynamics in exocytosis.In addition to the above physiological functions,Syt7 is involved in some pathophysiological processes,such as trypanosoma cruzi invasion cells,Parkinson's disease,and pulmonary fibrosis.In addition,studies have reported that Syt7 is required for Ca2+ to regulate the secretion of lysosomal vesicles,which can promote the migration of neutrophils by regulating vesicle secretion.Therefore,we want to know whether the internal mechanism of Syt7 can affect the function of tumor cells.There are no reports on the effects of Syt7 on tumor cell,especially hepatocellular carcinoma cells.ObjectiveTo elucidate the effect of Syt7 on the proliferation of hepatocellular carcinoma cells SMMC-7721 and BEL-7404 in vitro study,and to confirm the inhibition of Syt7 on the growth of xenografted tumor in nude mice in vivo experiment.Methods(1)We designed RNA interference target sequence and constructed target gene RNA interference lentiviral vector by using Syt7 gene as a template;Lentivirus containing Syt7 gene RNA interference sequence was used to infect hepatocellular carcinoma cells SMMC-7721 and BEL-7404,and it is divided into experimental group(shSyt7 group)and control group(shCtrl group);(2)qRT-PCR was used to detect the mRNA level of Syt7 in SMMC-7721 and BEL-7404 and to assess interference efficiency;Western Blot was used to detect the protein expression of Syt7;(3)Celigo cell counting was used to detect cell growth;(4)Cloning experiments was used to detect the ability of cells in forming tumors after Ientivirus interference;(5)Pl-FACS was used to detect changes of cell cycle;(6)MTT was used to detect changes of cell proliferation;(7)The growth of xenografted tumor in nude mice was used to detect the effect of interference with Syt7 on tumor growth inhibition;(8)Statistical analysis was performed using SPSS 21.0 software.All data were expressed as mean± standard deviation.Statistical analysis between groups of data was performed by t test.P<0.05 was considered statistically significantResults(l)Syt7-interfering lentiviral vector was successfully constructed,and a cell Iine stably interfering with Syt7 was established.Fluorescence observation showed that the cell infection efficiency was more than 70%;(2)The mRNA levels of Syt7 in the SMMC-7721 and BEL-7404 cells of the experimental group were significantly inhibited(P<0.05),and the knockdown efficiency was 62.3%and 52.3%respectively.Western Blot showed that there was a significant reduction in the exogenous protein expression of Syt7;(3)Celigo was continuously tested for 5 days,and the growth of SMMC-7721 and BEL-7404 cells in the experimental group was significantly inhibited,suggesting that Syt7 was significantly associated with the growth of SMMC-7721 and BEL-7404 cells;(4)Cloning experiments showed that the number of colonies in SMMC-7721 and BEL-7404 cells in the experimental group was decreased,suggesting that Syt7 was significantly associated with the clone ability of SMMC-7721 and BEL-7404 cells;(5)Cell cycle detected by PI-FACS.It was showed that the SMMC-7721 cells in the G1 phase were significantly reduced and the cells in the S phase were significantly increased in the experimental group,while there was no significant change in the G2/M phase.In addition,the BEL-7404 cells in the GI phase were significantly increased and the cells in the G2/M phase was significantly reduced in the experimental group,while there was no significant change in the S phase.It was suggested that Syt7 was significantly associated with the cell cycle distribution of SMMC-7721 and BEL-7404 cells;(6)The MTT assay was continuously tested for 5 days,and the proliferation rates of SMMC-7721 and BEL-7404 cells in the experimental group were significantly inhibited.It was suggested that Syt7 was significantly associated with the proliferation of SMMC-7721 and BEL-7404 cells;(7)The fluorescence expression was signiflcantly decreased,and the volume and weight of the tumor were significantly decreased after interfering with Syt7 in nude mice.Conclusions(l)Interfering with Syt7 inhibits proliferation of SMMC-7721 and BEL-7404 cells.(2)Interfering with Syt7 can induce cell cycle arrest in hepatocellular carcinoma cells.(3)Interfering with Syt7 can inhibit the clone formation of hepatocellular carcinoma cells.(4)Interfering with Syt7 can inhibit the growth of xenografted tumor in nude mice.BackgroundComprehensive treatment of hepatocellular carcinoma is diverse,and radical surgical resection is still the preferred treatment.However,due to the poor biological behavior of hepatocellular carcinoma,such as multi-center,rapid progress,high risk of recurrence and metastasis,the overall effect is poor and the survival rate is low.Exogenous factors such as environmental pollution,endogenous chromosomal changes,and intracellular genes changes eventually lead to malignant transformation of cells and tumorigenesis.Therefore,it is insufficient to show that some genes are abnormally expressed and are functional in hepatocellular carcinoma cells and tissue samples,To clarify the mechanism of occurrence and development of hepatocellular carcinoma,it is necessary to further explore the gene-related signal transduction pathway and to clarify the role of gene in the pathway.Based on this,it may be possible to find a target gene for targeted therapy of hepatocellular carcinoma.Many studies have shown the signaling pathways in hepatocellular carcinoma,such as Wnt/?-catenin signaling pathway,p38-MAPK pathway,Hedgehog signaling pathway,P13K-hkt,MAPK,SAPK/JNK,NF-KB,epithelial-mesenchymal transition,autophagy related pathways,apoptosis-related death signaling pathways,and mitochondrial pathways,all of which may play roles in hepatocellular carcinoma.The role of Syt7 has never been reported in tumors.Our previous studies have shown that Syt7 is highly expressed in tissue specimens of hepatocellular carcinoma and hepatocellular carcinoma cells.In vitro and in vivo experiments further show that Syt7 can promote hepatocellular carcinoma cell proliferation and progression,while the pathway of Syt7 involved in hepatocellular carcinoma remains unclear.This part seeks to clarify the signaling pathways of Syt7 in hepatocellular carcinoma and the role of Syt7 in the pathway,which may provide an experimental basis for finding new therapeutic targets for hepatocellular carcinoma.Objective To detect the changes of key signaling molecules in signaling pathway in hepatoma cell line SMMC-7721 after interference with Syt7.Methods(1)We designed RNA interference target sequence and constructed target gene RNA interference lentiviral vector by using Syt7 gene as a template;Lentivirus containing Syt7 gene RNA interference sequence was used to infect hepatocellular carcinoma cell SMMC-7721,and it is divided into experimental group(shSyt7 group)and control group(shCtrl group);(2)qRT-PCR was used to detect mRNA level of Syt7 in SMMC-7721,and to assess the interference efficiency;(3)The changes of key signaling molecules in the signaling pathway in the experimental group and control group were detected by Cell Signaling Technology(CST)PathScan(?)Stress and Apoptosis Signaling Antibody Array Kit;(4)The expressions of key proteins in SMMC-7721 cell were further verified by Westerm Blot after Syt7 interference(5)Statistical analysis was performed using SPSS 21.0 software.All data were expressed as mean ± standard deviation,P<0.05 was considered statistically significant.Results(1)Syt7-interfering lentiviral vector was successfully constructed,and a cell line stably interfering with Syt7 was established.Fluorescence observation showed that the cell infection efficiency reached more than 70%;(2)The mRNA expression level of Syt7 in the SMMC-7721 cells of the experimental group was inhibited(P<0.05),and the knockdown efficiency reached 53.4%(3)In SMMC-7721 cells,after RNA interference with Syt7,the related genes in the Stress and Apoptosis signaling pathway were detected,and the phosphorylation levels of p53 and Chkl proteins were significantly up-regulated in the pathway.(4)The expression of p53 and Chkl gene exogenous protein was up-regulated in SMMC-7721 cells after Syt7 interference.Conclusions Interference with the Syt7 can inhibit the proliferation of hepatoma cell SMMC-7721 and arrest the cell cycle in the S phase by activating the Chkl-p53 signaling pathway. |
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