| Objective: Multiple myeloma(MM)is the most common type of malignant plasma cell tumor.The malignant proliferation of monoclonal plasma cells,extensive infiltration and secretion of a large amount of monoclonal immunoglobulin(M protein)in MM causes extensive bone destruction,infection,anemia,hypercalcemia and renal insufficiency.In recent years,with the clinical application of new drugs and the widespread development of autologous stem cell transplantation,the remission rate and disease-free survival of newly diagnosed and refractory patients have improved significantly.However,MM is still an incurable disease.Therefore,we need to further explore the molecular mechanism of the occurrence and development of MM,find more effective diagnostic and therapeutic targets to provide important biological basis.PIWI interacting RNA(pi RNA)is a newly discovered nc RNA with a length of 21-35 nt.It usually functions in conjunction with the PIWI protein,the subfamily of Argonaute proteins,and is associated with silencing of transposons in the development of the germline.Although past research believes that pi RNA only plays a role in gonadal cells.However,recent studies have shown that pi RNAs are differentially expressed in a variety of tumors and effectively control the biological functions of different types of tumors,such as tumor cell proliferation,migration,invasion and differentiation,which may become potential biomarkers of tumors.In MM,there are few studies of pi RNA.At present,there is only one pi RNA,pi RNA-823,which is highly expressed in MM cells,and participates in the survival of MM cells by affecting the bone marrow microenvironment.Thus,we explored the differential expression and clinical significance of PIWIinteracting RNA-004800(pi R-004800)in bone marrow supernatants of MM patients and healthy donors.The biological functions and the related molecular biological mechanisms of pi RNA in MM cells were further explored.Methods: Part Ⅰ: Screening for differentially expressed pi RNAs in exosomes from bone marrow supernatants of MM patients and heakthy donors.1.Bone marrow supernatants from 3 MM patients and 3 healthy donors were collected,exosomes were extracted,and differentially expressed pi RNAs were screened by gene sequencing technology.2.Large sample verification: Exosomes were collected from bone marrow supernatant of 66 MM patients and 17 normal humans and cell culture supernatant,RNA was extracted,and differentially expressed pi RNAs were detected by RT-PCR to verify whether their expression trends were consistent with sequencing.RNA was extracted from CD138 + plasma cells of 33 MM patients,mononuclear cells of 18 normal humans and MM cell lines(RPMI8226,U266),and RT-PCR was used to detect the expression level of pi RNA in the cells for further verification.3.Based on the sequencing and verification results,pi R-004800 was selected for further research in this subject.The clinical information of MM patients was collected,and the relationship between the expression level of pi R-004800 and the clinical stage of ISS was clarified.Prat Ⅱ: Effects of pi R-004800 in MM cell lines(RPMI8226 and U266)on cell proliferation,apoptosis and autophagy 1.Antagomir-4800 and agomir-4800 were used to silence or up-regulate the expression of pi R-004800 in MM cell lines(RPMI8226 and U266).2.MM cells were divided into the experimental group(antagomir-4800 / agomir-4800 group),the control group(antagomir / agomir-NC group)and the blank group(nontransfected group).MTS method was used to detect the proliferation of three groups.The apoptotic levels of the three groups were detected by flow cytometry.Western blot was used to detect the expression of apoptosis-related proteins Bcl-2,Bcl-XL,BAD,cleaved PARP,cleaved caspase-3,Mcl-1,total caspase-3 and Stat3 in transfected cells.Western blot was used to detect the expression of autophagy markers LC3 B,p62 and becline1 in transfected cells.3.Tumor xenograft model was established.The effects of pi R-004800 on the proliferation,apoptosis and autophagy of MM cells were confirmed by in vivo experiments.Patr Ⅲ: Study on the regulatory mechanism of pi R-004800 in MM cells 1.S1 P receptor signaling pathway agonists S1 P and inhibitors FTY720 were used to treat RPMI8226 and U266 cells,respectively.Then RT-PCR was used to detect the expression of pi R-004800.2.Si RNA was used to down-regulate the expression of CDC42 in MM cell lines.RTPCR was used to detect CDC42 knockdown efficiency and the expression of pi R-004800 in MM.3.After transfection of MM cells with antigomir-4800 or antigomir-NC,the expressions of p-Akt,Akt,p-m TOR and m TOR were detected by Western blot.4.S1 P was used to treat antagomir-4800 or antagomir-NC transfected MM cells.Western blot was used to detect the expression of p-Akt,Akt,m TOR and p-m TOR.Results: Part Ⅰ: Screening results of differentially expressed pi RNAs in MM patients and healthy donors 1.Exosomal small RNA sequencing screened that 1521 pi RNAs were up-regulated and 498 pi RNAs were down-regulated in MM.2.In the screening results,4 pi RNAs(novel-pi R15,pi R-004800,novel-pi R334,pi R-016659)with the most significant difference in expression up-regulation were selected.RT-PCR results showed that pi R-004800 was up-regulated in the exosomes of bone marrow supernatant and CD138+ plasma cells of MM patients,MM cell lines and exosomes of MM cell lines.3.MM patients were divided into three groups according to the International Staging System(ISS): ISS Ⅰ,ISS Ⅱ,and ISS Ⅲ.In patients with ISS Ⅱ and ISS Ⅲ,the expression level of pi R-004800 was significantly higher than that in the ISS Ⅰ group and the normal control group(p<0.01).The results suggest that the expression level of pi R-004800 is positively correlated with the clinical stages of MM.Part Ⅱ: pi R-004800 plays an important role in the proliferation,apoptosis and autophagy of MM cell lines 1.We successfully prepared antagomir / agomir-4800 and antagomir / agomir-NC.Transfection of antagomir-4800 down-regulated the expression of pi R-004800 in MM cells(p<0.01),and transfection of agomir-4800 significantly increased the expression of pi R-004800 in MM cells(p<0.01).2.Compared with the NC group,the cell proliferation ability of the antigomir-4800 group was significantly reduced(p<0.01),and the cell proliferation ability of the agomir-4800 group was increased(p<0.01).3.Flow cytometry’s results showed that the early apoptotic rate of the antigomir-4800 group was significantly increased(p<0.05).4.The downregulation of pi R-004800 decreased the expression of anti-apoptotic proteins Bcl-2,Bcl-XL,Mcl-1 and total caspase-3;Conversely,this increased the expression of pro-apoptotic proteins BAD,cleaved PARP and cleaved caspase-3.5.The results of Western blot showed that LC3B-Ⅱ’s expression in antagomir-4800 group was up-regulated and p62’s expression was down-regulated,suggesting that downregulating pi R-004800 can activate autophagy in MM cells.MM cells with downregulation of pi R-004800 were treated with two autophagy inhibitors 3-methyladenine(3MA)and Bafilomycin A1.By MTS assay,it showed that autophagy inhibitors rescued cell death induced by pi R-004800 knockdown.6.Animal experiments showed that the growth of subcutaneous tumors in mice treated with antigomir-4800 was significantly inhibited.Immunohistochemical staining and Western blotting were performed on tumor tissue.The experimental results are consistent with the results of in vitro experiments.Part Ⅲ: The S1 P receptor signaling pathway can regulate the PI3 K / Akt / m TOR signaling pathway in MM cells through pi R-004800 1.After FTY720 treatment,the expression of pi R-004800 in MM cell lines was significantly reduced(p<0.05).After S1 P treatment,the pi RNA-004800 expression was significantly increased in MM cell lines(p<0.01).After CDC42 knockdown,the expression of pi R-004800 in MM cell lines was significantly reduced(p<0.01).2.Comparing with the NC group,the expression of p-Akt,Akt,m TOR and p-m TOR in antagomir-4800 group significantly decreased(p<0.01).3.We treated pi R-004800 downregulated MM cells with S1 P.We found that the expressions of Akt,p-Akt,m TOR and p-m TOR could be partly rescuedConclusion: 1.The expression of pi R-004800 in MM patients and cell line samples was significantly higher than that in normal people,and its expression level was positively correlated with the ISS stage of MM patients.2.pi R-004800 can promote the proliferation of MM cells.After down-regulation,pi R-004800 can inhibit MM cell proliferation in vivo and in vitro,promote MM cell apoptosis and induce autophagic death of MM cells.3.The S1 PR signaling pathway can regulate the expression of pi R-004800,and pi R-004800 can further affect the proliferation of MM cells by regulating the PI3K/Akt/m TOR signaling pathway. |
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