The Study Of PIWIL1 Promoting The Progression And Drug Resistance In Multiple Myeloma Through Modulating Autophagy

Objectives:1.To detect the relative expression level of PIWIL1 in CD138+ cells from Multiple Myeloma(MM)patients and MM cell line.2.To study the role of PIWIL1 in the growth and drug resistance of MM cells.3.To explore the effect of PIWIL1 on autophagy in MM cells and whether it promotes the drug resistance of MM cells through autophagy.Methods:1.CD138+ cells were isolated from bone marrow which were collected from12 MM patients and 4 healthy volunteers.The expression of PIWIL1 m RNA and protein was detected by q RT-PCR and Western Blot.2.(1)The expression of PIWIL1 m RNA and protein in MM cell lines was detected by q RT-PCR and Western Blot.(2)PIWIL1 knock-in MM cells were obtained by transfection of PIWIL1 c DNA lentivirus and PIWIL1 RNAi lentivirus was used to knock-down the PIWIL1 expression in MM cells.Correspondingly,scramble control lentivus was used as negative control.1)The efficiency of transfection was determined by the detection of PIWIL1 m RNA and protein expression.2)CCK-8 was used to evalute the effects of PIWIL1 in cell proliferation as well as drug resistance of MM cells.3)Flow cytometry analysis was used to evaluate the apoptosis in MM cells.4)The morphology and number of autophagosomes in MM cells were observed by transmission electron microscope.5)Western Blot was used to evaluate the expression of autophagy-related proteins including p AKT,m TOR,p-m TOR,P62,LC3A/B,Parkin,TBK-1,p-TBK-1,OPTN as well as apoptosis-related proteins such as BCL-2 and Caspase-3;6)The difference of the sensitivity of MM cells to Dexamethasone(Dex)with or without Cs A treatment was detected by CCK-8.3.In vivo experiment NOD/SCID mice were implanted subcutaneously with different group of myeloma cells and the development and growth of tumors were monitored for six weeks post implantation.We used immunohistochemistry to evaluate the expression of P62,p-m TOR,OPTN and BCL-2 in xenografted tumors.Results:1.The expression level of PIWIL1 was higher in the CD138+ cells and MM cell lines than that in the normal controls;2.CCK-8 results showed that the cell viability was significantly increased after the knock-in of PIWIL1,while the cell viability was decreased after knock-down of PIWIL1 expression.After the knock-in of PIWIL1,the sensitivity of MM cells to Dexamethasone?Bortezomib?Doxorubicin was decreased,the knock-down of PIWIL1 expression increased the sensitivity of MM cells to these drugs;3.Flow cytometry results showed that PIWIL1 had no significant effect on the apoptosis of MM cells.4.Transmission electron microscope showed that the number of autophagosomes were significantly increased after the knock-in of PIWIL15.Western Blot results showed that the expression of autophagy-related proteins such as LC3A/B-II,Parkin,p-TBK1,OPTN were significantly increased after the knock-in of PIWIL1,whereas the expression of m TOR,p-m TOR and P62 were decreased.Conversely,the expression of abovementioned proteins were oppositely regulated after knock-down the PIWIL1 expression.There is no significant difference in the expression of apoptosis-related proteins including BCL-2 and Caspase-3 between PIWIL1 knock-in MM cells,PIWIL1 knock-down MM cells,and negative control.6.CCK-8 results showed that there is no significant difference in the sensitivity of MM cells to Dexamethasone between PIWIL1 knock-in MM cells and negative control after inhibiting mitophagy.Samely between PIWIL1knock-down MM cells and negative control.7.Tumor mass in mice xenografted with PIWIL1 Knock-in MM cells was significantly enhanced with decreased expression of P62,p-m TOR,and increased OPTN expression.Conversely,the expressions of these proteins were oppositely regulated in mice xenografted with PIWIL1 knock-down MM cells.There is no significant difference in the expression of apoptosis-related protein BCL-2 between xenografted with PIWIL1 Knock-in MM cells,PIWIL1 knock-down MM cells,and negative control.Conclusions:1.PIWIL1 was highly expressed in CD138+ cells from MM patients and MM cell lines.2.PIWIL1 could promote the growth of MM cells both in vito and vivo.3.PIWIL1 could promote the growth and drug resistance of MM cells through mitophagy.