| Background: The occurrence and development of any cancer are related to the proliferation and apoptosis of cells.It is beneficial for the treatment of tumor only by inhibiting the proliferation and promoting the apoptosis of tumor cells.From the perspective of epidemiology,gastric cancer is one of the tumors with high incidence all over the world.Most patients with advanced gastric cancer have been found without valid clinical treatment.Therefore,early detection and diagnosis is particularly important.It has been accepted in public that obesity is one of the important factors leading to various tumors.Some studies have shown that gastric cancer is related to abnormal lipid metabolism,but the specific mechanism is rarely studied.PLIN2,or perilipin 2,is a lipid differentiation related protein,which mainly exists in various tissues and cells,and mainly facilitate the storage of lipid droplets in cells with phospholipids.Some studies have shown that the expression level of PLIN2 in gastric cancer tissues is significantly higher than that in adjacent tissues.The expression level of PLIN2 is also related to the stage and type of gastric cancer,but the specific biological function and mechanism of PLIN2 in gastric cancer cells have not been studied.Ferroptosis is a hot topic in recent years,which is essentially an intracellular lipid peroxidation stress reaction.More and more studies show that Ferroptosis plays an important role in the occurrence and development of cancer,but the correlation between Ferroptosis and gastric cancer is seldom mentioned.The role of PLIN2 in Ferroptosis of gastric cancer has not been studied.It is unknown whether PLIN2 can be used as a biomarker and a target of drug therapy to diagnose and treat gastric cancer.Therefore,it is very important for us to study the pathogenesis of gastric cancer with elevation of PLIN2.In this paper,we studied the relationship between PLIN2 and the biological function of human gastric cancer cell lines SGC7901 and MGC803 based on the modification of Ferroptosis and explained the mechanism of lipid metabolism disorder leading to gastric cancer.We speculated that PLIN2 might be a potential biomarker for diagnosis and treatment of gastric cancer.Four parts as follows:Part Ⅰ High and low expression of PLIN2 in gastric cancer cell lines SGC7901 and MGC803 Establishment and effect on cell functionMethod: 1.Construction of plasmid vector with high and low expression of PLIN2.2.High and low expression PLIN2 plasmid vectors were transfected into SGC7901 and MGC803 cell lines respectively.3.The expression of PLIN2 in SGC7901 and MGC803 cells was detected by Rtq PCR and Western blotting respectively.4.The proliferation,apoptosis,survival rate and ROS function of gastric cancer cell lines SGC7901 and MGC803 were detected by CCK8,flow cytometry,clone colony and fluorescence microscopy in situ cell morphology.Result: 1.PLIN2 plasmid vectors with high and low expression were successfully constructed.2.Stable gastric cancer cell line with high and low expression of PLIN2 was successfully constructed.3.The results of Rt-q PCR and Western blotting showed that the expression of PLIN2 in cells with over-expression gene PLIN2 was significantly higher than that in control group,and the expression of PLIN2 in cells with low PLIN2 gene knockdown was significantly lower than that in control group.4.The results of cell function showed that compared with the control group,the proliferative ability and survival rate of Ov PLIN2 cells were significantly increased,while the apoptotic ability and the content of reactive oxygen species(ROS)were significantly decreased.On the contrary,the proliferative ability and survival rate of Sh PLIN2 cells were significantly decreased,while the apoptotic ability and ROS content were significantly increased.Part Ⅱ Construction of subcutaneous xenografts in vivo and verification of PLIN2 expression in nude miceMethod: 1.SGC7901 cells with over-expression and low-expression of PLIN2 gene were injected subcutaneously into five-week-old nude mice(BALB/c nu/nu)to measure the tumor volume,body length and weight of mice.The tumors were removed and the weight of tumors was compared after four weeks.2.The levels of PLIN2 in mouse tumors were detected by immunohistochemistry,immunoblotting,Rt-q PCR and HE staining.Result: 1.The model of subcutaneous gastric cancer in nude mice was successfully constructed.The tumor volume and weight、mouse weight with over-expression of PLIN2 cells were significantly higher than those of control group,while the tumor volume and weight 、 mouse weight with low expression of PLIN2 cells were significantly lower than those of control group.2.The level of PLIN2 in tumor tissue of over-expression gene PLIN2 mice was significantly higher than that of control group,while the level of PLIN2 in tumor tissue of low-expression gene PLIN2 mice was significantly lower than that of control group.Part Ⅲ RNA-seq Sequencing of Mouse Tumor Tissues and Biological Analysis of Differential GenesMethod: 1.Three groups of xenograft tumors were sent to BGI of Beijing Genome Research Institute.Total RNA was extracted by magnetic bead method and sequenced on BGI seq-500 system.2.Quantitative analysis of genes,analysis based on gene expression level(principal component,correlation,screening of differentially expressed genes,etc.)and further digging of differentially expressed genes among selected samples,such as enrichment analysis of GO functional significance,enrichment analysis of pathway significance,clustering,protein-protein interaction network and transcription factors,excavation analysis.Result: 1.Compare the number of different genes regulated by PLIN2 gene in each two groups of samples,and the volcanic map showed a total of 1138 differential genes were detected between NC and Ov PLIN2,including 120 up-regulated genes and 1018 down-regulated genes;627 down-regulated genes were detected between NC and Sh PLIN2,including 282 up-regulated genes and 345 down-regulated genes;1516 differential genes were detected between Sh PLIN2 and Ov PLIN2,including 1184 upregulated genes and 332 down-regulated genes.2.Venn map showed 86 common differential genes in NC vs Ov PLIN2,NC vs Sh PLIN2 and Sh PLIN2 vs Ov PLIN2.3.Cluster analysis showed the obviously differential gene expression of three group of samples.4.The analysis of GO showed the most significant GO function is lipid metabolism among three group of samples.5.The analysis of KEGG pathway showed the most significant difference was Ferroptosis pathway contains genes ACSL3,ALOX15,LC3 A and PRDM11,IPO7,which were potentially regulated by PLIN2.Part Ⅳ The mechanism of PLIN2 in promotion of gastric cancerMethod: 1.The important proteins related in Ferroptosis pathway regulated by PLIN2 were identified by Rt-q PCR,immunoblotting and immunohistochemistry.2.The size and number of lipid droplets in overexpressed and lowexpressed PLIN2 cells,the content of triglycerides in tumor tissues and were detected by Nile Red fluorescence staining and microanalysis respectively.Lee’s index of mice was evaluated among three groups.Result: 1.Overexpression of PLIN2 down-regulated the expression of ACSL3,ALOX15,LC3 A,and PRDM11 in Ferroptosis pathway but up-regulated IPO7 expression.When PLIN2 was knocked down,the expression of ACSL3,ALOX15,LC3 A,PRDM11 increases and the expression of IPO7 decreases.This result has been verified by Rt-q PCR,Western blotting and immunohistochemistry.2.According to the Nile Red fluorescence intensity,the lipid droplets in the cells with overexpressing PLIN2 were larger and more,while the lipid droplets in the cells with lowexpressing PLIN2 were smaller and fewer.The content of triglyceride in tumor tissue of high expression PLIN2 mice increased significantly,while that of low expression PLIN2 mice decreased significantly.Lee’s index evaluation showed that the body size of mice with high expression of PLIN2 was significantly larger than that of mice with low expression of PLIN2.To sum up,our study showed that PLIN2 can affect the proliferation and apoptosis of gastric cancer cells by regulating the important genes in Ferroptosis pathway in vivo and in vitro.Meanwhile,it also explained one of the reasons why abnormal lipid metabolism can lead to gastric cancer.Therefore,it probably not only be a biomarker for diagnosis,but also a new target for treatment. |
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