| Objective: The study aims to find the role of RASSF10 in the pathogenesis of multiple myeloma by investing the methylation status of Ras-related domain family 10 gene promoter in multiple myeloma and the relationship between RASSF10 and MM.Content:To study the expression level of RASSF10 gene and the methylation of RASSF10 promoter in MM cell lines and MM patients and their correlations.To detect the effect of RASSF10 overexpression on the proliferation and apoptosis of MM cells lines.To detect the role of RASSF10 in the tumor formation of myeloma cells in nude mice.Methods:1.The study enrolled 47 patients with MM and 19 normal controls in our hospital from June 2016 to August 2017.Among those patients,30 patients were newly diagnosed and relapsed MM,17 patients had remission to VGPR after treatment.Immunomagnetic beads were used to sort CD138+ cells in the bone marrow.Flow cytometry was used to measure the purity of CD138+ cells.The human multiple myeloma cell lines RPMI-8226,OPM-2,and U266 were used for cell culture.The expression of RASSF10 mRNA in CD138+ cells and MM cell lines were detected by qRT-PCR.The differences of RASSF10 mRNA expression levels were compared among newly diagnosed group and relapsed MM patients group,as well as the remission patients group and normal control group.The relationship of the expression level of RASSF10 mRNA and the overall survival of patients were analyzed.The comparison of the expression of RASSF10 gene had been made between myeloma cell lines(RPMI-8226,OPM-2 and U266)and the control group.2.RASSF10 was over-expressed in MM cell line by lentiviral transfection.The efficiency of lentiviral transfection was detected by flow cytometry,and the expression of RASSF10 mRNA was detected by qRT-PCR before and after transfection of RASSF10 with MM cell line.The Western Blot method was used to detect the expression of RASSF10 protein.CCK-8 was used to detect the proliferation of MM cell line before and after transfection.The flow cytometry was used to detect apoptosis of MM cell line before and after the transfection.The expression of apoptotic pathway protein was detected by Western Blot.The MM cell line methylation situation was detected by MSP method.After being treated of MM cells with 5-aza-2'-deoxycytidine,the expression of RASSF10 mRNA in MM cell lines was detected the level of change by qRT-PCR.3.The nude mice were used for tumorigenesis after overexpression of RASSF10.The effect of RASSF10-overexpressing MM cell line on the subcutaneous tumorigenesis in nude mice was compared.The tumor tissue was detected by pathological examination.The morphology of tumor cells was detected by HE staining.Myeloma cells and the expression of RASSF10 were verified by immunohistochemistry.Result:1.The purity of CD138+ cells sorted by bone marrow mononuclear cells was more than 90%.The expression levels of RASSF10 mRNA in CD138+ cells in the newly diagnosed and relapsed MM,remission,and normal control groups were(0.20±0.29),(0.64±0.61),and(0.62±0.61),respectively.There was statistical significance between the three groups(p<0.01).The expression of RASSF10 mRNA in the newly diagnosed and relapsed MM group was significantly lower than that in the remission group and the normal control group(p<0.01).There was no significant difference in the expression of RASSF10 mRNA between the MM remission group and the normal control group(p>0.05).The RASSF10 mRNA expression level in newly diagnosed and relapsed MM was delimited by 0.1,followed up for 15 months(95% CI 11-18).The OS of RASSF10 mRNA expression<0.1 MM group was 10 months(95% CI 6-14),RASSF10 mRNA expression> 0.1 MM group was 16 months(95% CI 12-20).The expression levels of RASSF10 mRNA in the MM cell lines RPMI-8226,OPM-2,and U266 were significantly decreased than those in the normal control group.2.The transfection efficiencies rates of RASSF10 in multiple myeloma cell lines were 69.3% for RPMI-8226,58.67% for OPM-2,and 34.7% for U266.Select RPMI-8226 and OPM-2 for follow-up experiments.The relative expression levels of RASSF10 mRNA before and after transfection in MM cell line RPMI-8226 were(7.83e-5±1.01e-5),(5.81e-2±2.72e-3).There was statistical significance between the two groups(p<0.01).And the relative expression of RASSF10 mRNA before and after transfection in MM cell line OPM-2 were(1.05e-5±1.77e-6),(6.16e-2±1.47e-2),respectively.There were statistical significance between the two groups(p<0.01).After overexpression RASSF10 in RPMI-8226 and OPM-2,the OD values of the two cell lines were decreased to have a more and more significant difference from that of control group as time went on.The OD values of RPMI-8226 at 24 h,48 h,72 h,and 96 h before and after transfection were(0.94±0.01),(1.67±0.06),(1.81±0.06),(2.17±0.08)and(0.98±0.03),(1.27±0.08),(1.28±0.05),(1.35±0.02);OPM-2 were(1.34±0.03),(1.68±0.10),(1.84±0.03),(2.38±0.05)and(1.34±0.03),(1.50±0.04),(1.62±0.08),(1.99±0.01).Among these date,there were statistically significant in 48 h,72h,and 96h(p<0.01).After overexpression RASSF10 in RPMI-8226 and OPM-2,the apoptosis rate of the two cell lines were increased to have a more and more significant difference from that of control group as time went on.The apoptosis rates of RPMI-8226 at 24 h,48h,72 h,96h before and after transfection were(27.37±2.67)%,(39.42±3.38)%,(40.97±1.73)%,(47.99±2.62)% and(41.02±4.63)%,(44.76±5.07)%,(52.31±1.23)%,(63.96±8.19)%;OPM-2 were(17.60±0.99)%,(19.12±2.23)%,(20.25±1.14)%,(24.70±0.78)% and(32.47±2.55)%,(35.26±7.03)%,(33.38±1.72)%,(44.04±1.72)%.Among these date,there were statistically significant between 24 h,72h and 96h(p<0.01).The protein expression of RPMI-8226 and OPM-2 in the MM cell line with restored expression of RASSF10 was detected by Western blot at the protein level.The expression of apoptosis-related protein caspase-3 was up-regulated,while bcl-2,a apoptosis inhibitor,was down-regulated at the protein level.The methylation of RPMI-8226,OPM-2,and U266 promoters in three MM cell lines was detected by methylation-specific PCR.The results showed that the RASSF10 gene promoter in MM cell lines RPMI-8226,OPM-2,and U266 were all hypermethylated.The MM cell lines RPMI-8226,OPM-2,U266 were treated with 5-aza-2'-deoxycytidine.The expression levels of RASSF10 before and after demethylation treatment in MM cell lines RPMI-8226,OPM-2,U266 were RPMI-8226(1.57e-5±2.02e-6)and(1.44e-3±7.79e-4),and OPM-2(2.95e-5±1.07e-5)and(3.92e-3±1.91e-3),U266(1.53e-6±5.41e-7)and(3.67e-5±1.52e-5),and there was a statistically significant difference between before and after demethylation treatment(p < 0.05).3.In animal experiments,Nude mice(BALB/c-nu)were injected subcutaneously with the MM cell lines RPMI-8226 and OPM-2 in the right scapular region.The OPM-2 nude mice had subcutaneous tumor formation.The lymph nodes,liver and spleen of the mice were significantly enlarged,and myeloma cells infiltrated in the liver.After RASSF10 expression restored,both volume and the mass of the subcutaneous tumor decreased.The tumor volume and mass of the OPM-2 control group were(96.97±15.22)mm3 and(87.57±19.75)mg respectively.The tumor volume and mass of OPM-2 over-expressing RASSF10 were significantly reduced.They were(16.56±3.15)mm3 and(19.90±4.60)mg,respectively.There was a statistically significant difference between the two groups(p<0.01).The tumor tissue formed subcutaneously in nude mice was stained with HE and showed a large number of irregular cells,short spindle or cubic,irregular nuclear,coarse staining,and obvious nucleoli;Immunohistochemistry: CD138+ in OMP-2 control group is about 40-50 %,RASSF10 is approximately 30%;CD138+ in OPM-2 which RASSF10 gene expression is restored is approximately 10-20%,and RASSF10 is approximately 50-60%.Conclusion:1.The expression level of RASSF10 gene decreased in MM patients with newly diagnosed and relapsed patients.The level was restored after the remission of the MM patients.Companing to the normal control group,there was no difference on the RASSF10 gene expression level.It shows that the expression level of RASSF10 gene is related to the pathogenesis of MM.The overall survival of patients with significantly lower RASSF10 gene expression in both newly diagnosed and relapsed MM patients were lower than that of MM patients with relatively higher RASSF10 gene expression.The expression levels of RASSF10 in the MM cell lines RPMI-8226,OPM-2,and U266 were significantly lower than those in the normal control group,demonstrating that the expression level of RASSF10 gene decreased in MM patients and MM cell lines.2.After the lentiviral transfection of RASSF10 in multiple myeloma cell lines RPMI-8226 and OPM-2,the expression of RASSF10 in both cell lines were restored,which proves that the transfection is effective.After the overexpression of RASSF10,the proliferations of the two cell lines were decreased with the apoptosis proportion increased.The expression of apoptosis-related protein caspase-3 was up-regulated,while bcl-2,a apoptosis inhibitor,was down-regulated at the protein level.These results indicate that RASSF10 can inhibit the proliferation of the MM cells as well as promote the apoptosis.Methylation-specific PCR was used to detect the methylation status of promoters of RPMI-8226,OPM-2,and U266 genes in three MM cell lines.The results showed that these MM cell lines were hypermethylated,which indicates that the RASSF10 gene promoter in the MM cell line had methylation.After being treated with 5-aza-2'-deoxycytidine,the expression of RASSF10 in the three cell lines restored.The result showed that the decreased expression of RASSF10 mRNA was related to hypermethylation of RASSF10 gene promoter,and demethylation drugs could reverse gene silencing.3.The MM cell line OPM-2 had made obvious subcutaneous tumor formation in nude mice(BALB/c-nu).The lymph nodes,liver,and spleen of the mice were significantly enlarged.The myeloma cells were seen infiltrated in the liver.After RASSF10 expression restored,both volume and the mass of the subcutaneous tumor decreased.Histological examations had proven the tumor tissue to be myeloma cells.The increase of RASSF10 gene expression leading to reduced tumor formation in nude mice.Animal experiments confirmed that RASSF10 had the inhibitory effect on the growth of multiple myeloma cells. |
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