Study On The Characteristics Of Side Population Cells And The Significance Of ABCG2 In Multiple Myeloma

Objective: Multiple myeloma(multiple myeloma,MM)is a malignant plasma cell disease,the incidence rate of tumor incidence in Department of Hematology accounted for more than 10%,the current treatment methods contain combination chemotherapy,targeted therapy and hematopoietic stem cell transplantation,but due to the side effects of chemotherapy,patients life quality was unsatisfactoried.In the development of tumor treatment,drug resistance is still the obstacle,a "pump" exists in the cell membrane can pump out of chemotherapy drug.ABCG2 was first found in the drug resistance of breast cancer cell lines.Later,ABCG2 were detected in a variety of tumors.At present,ABCG2 has become a hot topic in the field of drug resistance research.ABCG2 function in multiple myeloma has not been clearly explained,what biological functions does ABCG2 play in multiple myeloma?Is it related to the progression of the disease?How about it for prognosis?How does ABCG2 associated with PTEN?Can ABCG2 play a protective role in the pathogenesis of multiple myeloma?In this paper,we will use Hoechst33342 staining to sort side population cells,and calculate the ratio of SP for each cell line,purpose is to identify and study the characteristics of stem cells of multiple myeloma cells and side population,study on the mutual regulation relationship between the side population cells and related signal pathway.We hope that this study can be used to target the killing of SP cells,and may be possible to explore new therapeutic strategies to overcome the resistance of traditional chemotherapy and improve the prognosis of patients with multiple myeloma.We used Lentivirus to improve the expression of ABCG2 to establish a simulation model of side population cells,and the application of PI3K/AKT pathway inhibitors for myeloma cell line analysis of expression changes of the expression of SP cells and related pathways before and after ABCG2.Next,we observed the expression changes before and after ABCG2 multiple myeloma cells in response to drugs and PTEN/PI3K/AKT signaling pathway,to study the mechanism of multiple myeloma drug resistance,and try to find new therapeutic targets and to improve the therapeutic effect and prognosis of multiple myeloma.We used Si RNA technology to silence ABCG2,to further understand the biological function of ABCG2 in PTEN/PI3K/AKT signaling pathway and SP cells.We need to solve the problem that how does ABCG2 participate in multiple myeloma we hope to find the feasible solution to the resistance reversal of multiple myeloma.Methods: 1.The side population cells of multiple myeloma cell lines were sorted by flow cytometry.2.Compare the side population cells and main population cells biological characteristics including cell cycle(PI staining),cell proliferation(CCK8),colony forming ability(soft agar assay),and drug resistance,and to detection the protein related to drug resistance.3.Overexpression of ABCG2 was induced by lentivirus infection in multiple myeloma cells.4.The ratio of SP cells and the sensitivity to chemotherapeutic drugs were compared before and after the overexpression of ABCG2,and the activity of PTEN/PI3K/AKT signaling pathway was detected by Real-Time PCR and Western blot.5.The proportion of SP cells,the sensitivity of cells to chemotherapeutic drugs and the activity of PTEN/PI3K/AKT signaling pathway were detected when cells were dealed with PI3K/AKT pathway inhibitor.6.The expression of ABCG2 was silenced when multiple myeloma cells were treated with Si RNA interference.7.Comparison of SP cell ratio,cell proliferation rate,cell sensitivity to chemotherapeutic drugs,and PTEN/PI3K/AKT signal pathway activity before and after interference.8.Select the multiple myeloma patients(30 cases),we use flow cytometry to sort SP cells,calculate the proportion of SP cells in clinical patients,and analyze the relationship between SP cells and clinical information.9.MM patients were grouped according to DS stage,and the relationship between PTEN,ABCG2,SP cell ratio,PI3K/AKT signaling pathway with disease progression was detected.Result:1.It is sure that multiple myeloma cell lines contains SP cells,and the proportion of SP cells in different cell lines was different.The SP proportion of NCI-H929 and U266 cell lines was 1.79±0.091% and 0.4623±0.0095%,respectively.2.To detect the cell cycle,it was found that compared with MP cells,most of SP cells were in the G0/G1 phase,most of them were in relatively quiescent phase;CCK8 method was used to detect the proliferation ability of SP and MP cells.It was found that SP cells had a rapid proliferation rate;Soft agar colony formation assay showed that SP cells had stronger colony forming ability,and the number of colonies was significantly higher than that of MP cells;Application of CCK8 method to detect the sensitivity of SP and MP cells on multiple myeloma chemotherapy drugs,found that sensitive of SP cells at the same concentration of drug was significantly lower than that of MP cells,SP cells possess more stronger capacity to fight the chemotherapy.Real-Time PCR detected the expression of ABC family members,and found that the expression of ABC family members in SP cells was significantly higher than that of MP cells,especially ABCG2,SP cells were about 10 times higher than MP cells.3.The Lentivirus infected cells lead to higher expression of ABCG2 than the uninfected cells.4.At the Lentivirus infected cells,m RNA and protein of ABCG2 expression increased significantly,the response of cells to chemotherapeutic drugs significantly decreased,PTEN protein expression decreased,and the activity of the PI3K/AKT pathway increased,the proportion of SP cells increased significantly.5.At the Lentivirus infected cells meet the inhibitor of PI3K/AKT pathway,PI3K/AKT pathway activity was significantly reduced,PTEN protein expression was increased,SP cell ratio was decreased.After the addition of the inhibitor,the response of the cells to the drug was enhanced to some extent.6.Si RNA interference can down regulate the expression of ABCG2 in m RNA and protein.7.After Si RNA interference,the proliferation of multiple myeloma cells decreased slightly,the expression of PTEN was increased,the activity of PI3K/AKT pathway was inhibited,and the proportion of SP cells was decreased.8.SP cells were detected in 30 cases of multiple myeloma patients,the proportion of SP cells varied from person to person range from 0.11% to 3.73%,we found that the proportion of SP cells has no relation with the patients age,sex,ISS stage,but associated with DS stage.9.We divided the multiple myeloma patients into different group according to DS staging,with the progress of the disease,the proportion of SP cells was increased,the level of PTEN m RNA was decreased,and the level of ABCG2 m RNA was increased.We detected the protein expression by Western blot,we found that the protein expression of ABCG2 increased with the progression of disease,and the activity of PI3K/AKT pathway also increased with the progression of disease.Conclusion:1.In the multiple myeloma cell lines,there are side population cells,and the SP cells of MM have the same biological characteristics with tumor stem cells.2.In multiple myeloma,overexpression of the ABCG2 negative feedback regulation of PTEN,upregulation of PI3K/AKT pathway activity and SP ratio,but the drug sensitivity decreased,while the inhibitor can counteract the protective effect brought by ABCG2.3.In multiple myeloma,ABCG2 down-regulation can negatively regulate the expression of PTEN,the PI3K/AKT signaling pathway is inhibited,the ratio of SP decreased,and the response to drug is increased.4.There were side population cells in patients with multiple myeloma,and the proportion of SP cells was not associated with age,sex,ISS stage,but with DS stage.With the progression of DS stage,the proportion of SP cells increased,suggesting that the proportion of SP cells was associated with disease progression.5.PTEN expression was decreased and ABCG2 expression increased in multiple myeloma patients.With the progress of the disease,PTEN decreased,ABCG2 increased,PI3/AKT activity increased,and the proportion of SP increased.6.ABCG2 has a negative feedback to the expression of PTEN in multiple myeloma,and plays an important role in the proportion of SP cells through the PI3K/AKT pathway.