| Background and objectives:Gastric cancer is one of the principal malignant tumors at present.Although great progress of prevention and treatment of gastric cancer in China in recent years has changed tremendously,the 5-year survival rate remains still very low.The reason is that the specific mechanism of the occurrence and development of gastric cancer are not clear.Gastric cancer stem cells are some cells with stem cell characteristics,which are highly associated with malignancy,drug resistance and metastasis of tumors.SPOP gene is a highly conserved gene,and its deletion may lead to the occurrence and development of tumors.Previous studies have found that SPOP plays an important role in many solid tumors,but its role in gastric cancer stem cells and other stem cells remains unclear.In addition,SPOP is down-regulated in gastric cancer.It is well known that there are many mechanisms involved in the protein expression process.In the process of tumorigenesis and development,microRNAs play an important role in the regulation of cell proliferation,differentiation,and apoptosis.However,it is difficult to see how cells are regulated.The purpose of this study is to explore the expression and function of SPOP in gastric cancer and related pathways of targeted regulation of miR-543,so as to provide some new ideas for the treatment of gastric cancer.Methods:1.Gastric cancer cell line MKN-45 was cultured and separated by suspension culture.Gastric cancer stem cells were identified by immunofluorescence and Western blot using stem cell markers.The tumorigenicity of gastric cancer stem cells was tested by animal experiments.2.Western blot and real-time quantitative PCR were used to determine the expression of SPOP in gastric cancer and adjacent normal tissues.3.To analyze the relationship between SPOP expression and clinicopathological characteristics in gastric cancer.4.To detect the expression of SPOP in MKN-45 Spheroid cells and adherent cells.5.The expression of SPOP in tumor stem cell markers(Oct3/4,Sox2,and CD44)and the corresponding sphere formation ability were detected by Western blot.6.overexpressing SPOP Spheroid cells and control cells were inoculated into nude mice to observe the tumorigenesis.7.36 pairs of gastric cancer and adjacent tissues were used to detect the expression of miR-543 and SPOP by real-time quantitative PCR.Then the correlation coefficient of Spearman was utilized to analyze the data,and the correlation between the expression of miR-543 and SPOP in gastric cancer was obtained.8.Luciferase reporter gene assay confirmed whether SPOP is a direct target of miR-543 in gastric cancer.9.By real-time quantitative PCR and Western blot analysis,the mRNA and protein expression of miR-543 and SPOP in MKN-45 and AGS cells with transfecting of miR-543 mimic group was compared to that in the control group.And the mRNA and protein expression of miR-543 and SPOP in MKN-45 and AGS cells with transfecting of miR-543 inhibitor group was compared to that in the control group.10.Cell migration and invasion were observed after transfection of miR-543 mimic,miR-543 inhibitor,and miR-543 inhibitor+SPOP siRNA.11.Western blot was used to detect the expression levels of SPOP,E-cadherin and vimentin in MKN-45 and AGS cells transfected with miR-543 mimic and miR-543 inhibitor,indicating the role of EMT in this process.Results:1.Successfully cultured and separated suspended spheroid cells,with the passage of time,the number of spheroid cells gradually increased,spheroids continue to grow.2.Immunofluorescence,Western blot,and real-time quantitative PCR were utilized to analyze the differences in the expression of stem cell markers CD44,Sox2 and Oct3/4 between suspended spheroid cells and adherent cells.It was found that suspended spheroid cells possessed the characteristics of cancer stem cells,and their globular ability and expression of stem cell markers were significantly higher than those of adherent cells,and their tumorigenicity was also markedly enhanced.3.Western blot and real-time quantitative PCR were utilized to detect the expression of SPOP in gastric cancer and adjacent normal tissues.The results showed that the expression levels of SPOP protein and SPOP mRNA in gastric cancer tissues were significantly lower than that in corresponding adjacent tissues,which played an anti-oncogene role.4.Analyzing the relationship between SPOP expression and clinicopathological characteristics in 60 cases of gastric cancer,we found that SPOP expression in gastric cancer was related to TNM stage(P=0.038),differentiation degree(P=0.029),depth of invasion(P=0.009),lymph node metastasis(P=0.001),and the difference was significant,but not significant with gender,age,tumor size,and distant metastasis.Cox regression was utilized to analyze the expression of SPOP,TNM stage,depth of invasion,lymph node metastasis,distant metastasis,sex,age,location and differentiation of tumors and the survival time of gastric cancer patients.The results showed that SPOP expression(P<0.001),TNM stage(P<0.001),depth of invasion(P=0.007),lymph node metastasis(P=0.034)and differentiation degree.(P=0.041)is a factor affecting the survival time of patients with gastric cancer.The difference has statistical significance.By introducing these significant factors into the Cox proportional risk model,we found that SPOP expression(P=0.002),TNM stage(P=0.018)and differentiation(P=0.010)were independent factors affecting the survival time of gastric cancer.The kaplan-Meier method was used to analyze the relationship between SPOP expression and survival time of gastric cancer patients.The results showed that the survival time of patients with high expression of SPOP was markedly different from that of patients with low expression.The 5-year survival rate of patients with high expression of SPOP was higher than that of patients with low expression(P=0.036).5.By transfecting SPOP-overexpressing lentivirus into sphere cells,that is,adding SPOP-overexpressing lentivirus-transfecting sphere cells into fresh culture medium,and comparing the sphere cells treated with no-load lentivirus with the sphere cells treated with no-load lentivirus,we observed and compared the formation of progeny sphere cells after transfection of SPOP 14 days,and calculated the clonal sphere formation rate of the two groups of cells.The results showed that the expression of SPOP protein in gastric cancer stem cells was significantly lower than that in adherent cells(P<0.05),and the expression of SPOP gene in gastric cancer stem cells was also significantly lower than that in adherent cells(P<0.05).6.By transfecting SPOP lentivirus into sphere cells,it was found that over-expression of SPOP could significantly inhibit the expression of tumor stem cell markers(Oct3/4,Sox2,and CD44),and the sphere formation rate was also significantly inhibited.Spheroid cells overexpressing SPOP and control cells were inoculated into nude mice respectively.It was found that overexpressing SPOP could significantly inhibit the growth of tumors(P<0.05).7.We selected 36 pairs of gastric cancer tissues and adjacent tissues to detect the expression of miR-543 and SPOP by real-time quantitative PCR.The expression of miR-543 in cancer tissues was stronger than that in adjacent normal tissues(P<0.001).The expression of SPOP was consistent in cancer tissues(P<0.001).The correlation coefficient of Spearman was utilized to analyze the data.It was found that the expression of miR-543 in gastric cancer tissue was negatively correlated with the expression of SPOP.8.Luciferase reporter gene assay confirmed that SPOP is a direct target of miR-543 in gastric cancer.9.Transfection of miR-543 mimic into MKN-45 and AGS cells by qPCR and Western blot dramatically reduced the expression of SPOP mRNA and protein(P<0.05).After transfection of miR-543 inhibitor,the levels of SPOP mRNA and SPOP protein in MKN45 and AGS cells upregulated significantly(P<0.05).10.By transfecting miR-543 mimic and inhibitor,we can conclude that miR-543 has an effect on the migration and invasion of gastric cancer cells.Western blot analysis showed that the expression of SPOP and adhesion decreased and the level of vimentin increased in the cells transfected with miR-543 mimic,while the result of transfected with miR-543 inhibitor was opposite.Therefore,it is suggested that miR-543 induces EMT in gastric cancer cells.Conclusions:1.Spherical cells cultured by suspension culture have the characteristics of cancer stem cells and have stronger tumorigenicity than adherent cells.2.SPOP is an anti-oncogene in gastric cancer and is more expressed in gastric cancer globular cells than in adherent cells.SPOP can serve as an independent factor for survival time of gastric cancer.3.miR-543 plays a promotive role in gastric cancer and has a negative correlation with target SPOP,and EMT has been proved to play an important role in its regulation process. |
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