Effect of TRIM31 Gene Silencing on the Proliferation and Apoptosis of U266 Cells and Its Mechanism / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of the tripartite motif containing 31 (TRIM31) gene silencing on the proliferation and apoptosis of multiple myeloma cells and its possible mechanism.@*METHODS@#The normal bone marrow plasma cells (nPCs) were selected as control, and the mRNA and protein expression levels of TRIM31 in human multiple myeloma cell lines (U266, RPMI-8226, NCI-H929 and KMS-11) were detected by RT-qPCR and Western blot. Recombinant lentivirol vector containing shRNA-TRIM31 and its negative control were used to infect U266 cells respectively, and the mRNA expression level of TRIM31 in infected cells was detected by RT-qPCR. Then cell proliferation, colony forming and apoptosis were analyzed by CCK-8, soft agar assay, and flow cytometry, respectively. The protein expression levels of TRIM31, cleaved-caspase-3, BCL-2, Bax, p-Akt (Ser473), Akt and PI3K (p110α) were evaluated by Western blot. In addition, the PI3K/Akt signaling pathway-specific inhibitor LY294002 and TRIM31-shRNA lentivirus were used to interfere with U266 cells, and the cell proliferation, apoptosis, and protein expression of p-Akt (Ser473) and Akt were detected by CCK-8, flow cytometry and Western blot, respectively.@*RESULTS@#Compared with nPCs, the expression levels of TRIM31 mRNA and protein in U266, RPMI-8226, NCI-H929 and KMS-11 cells were significantly increased (P<0.001), especially in U266 cells. After lentivirus infection, the levels of TRIM31 mRNA and protein in U266 cells were significantly decreased (P<0.001). TRIM31 silencing significantly inhibited the proliferation of U266 cells (P<0.05), attenuated the ability of cell cloning, improved cell apoptosis, up-regulated the protein expressions of cleaved-caspase-3 and Bas as well as down-regulated expressions of BCL-2, p-Akt (Ser473) and PI3K (p110α). There was no significant effect on Akt protein. Intervention of LY294002 significantly enhanced the inhibition on cell proliferation and the promotion on apoptosis mediated by TRIM31 gene silencing in U266 cells.@*CONCLUSION@#TRIM31 gene silencing can inhibit U266 cell proliferation and promote its apoptosis, which may be closely related to inhibition of PI3K/Akt signaling pathway.

Inhibitory Effect of Eukaryotic Expression Vector Bearing TFPI-2 Gene on SHI-1 Cell Growth / 中国实验血液学杂志

OBJECTIVE@#To construct a eukaryotic expression vector of human tissue factor pathway inhibitor-2 (TFPI-2) and to investigate the effect of TFPI-2 gene on the growth of acute monocytic leukemia cell line (SHI-1).@*METHODS@#The cDNA of TFPI-2 was obtained by genetic chemical synthesis, the TFPI-2 gene and the linear vector fragment were ligated and inserted into the multiple cloning site of PEGFP-N1 vector, and the eukaryotic expression vector PEGFP-N1-TFPI-2 was transfected SHI-1 cells, then the obtained SHI-1 cells was observed by fluorescence microscopy; MTT assay was used to detect the effect of TFPI-2 gene on the relative growth rate of SHI-1 cells at the different time-point; RT-PCR was used to detect TFPI-2 mRNA expression levels in the cells of each group before and after TFPI-2 transfection; TFPI-2 protein expression was detected by Western blot. The cells which successfully transfected with PEGFP-N1-TFPI-2 vector were named as SHI-1-TFPI-2 (experimental group), and the cells transfected with the empty vector pEGFP-N1 and the untransfected cells were named as SHI-1-V and SHI-1-P and used as the control group.@*RESULTS@#The human TFPI-2 gene eukaryotic expression vector PEGFP-N1-TFPI-2 was successfully constructed, then the transfected into SHI-1 cells, observed by fluorescence microscopy 24 hours later, as a result, the PEGFP-N1-TFPI-2 was successfully transferred into SHI-1 cells, and the number of fluorescent cells increased after 48 h and 72 h. RT-PCR showed that the gray scale ratio of TFPI-2 gene to β- actin in the experimental group was higher than that in the control group. The gray scale ratio was 0.51±0.04 in SHI-1-V group, 0.52±0.03 in SHI-1-P group, 0.87±0.08 in SHI-1-TFPI-2 group, and the difference between SHI-1-TFPI-2 and SHI-1-V, SHI-1-P group was statistically significant (P＜0.05).@*CONCLUSION@#The expression of TFPI-2 gene in PEGFP-N1-TFPI-2 can inhibit the growth of SHI-1 cells, which provides a research direction for gene therapy of leukemia in the future.

Inhibitory Effect of Serum Containing Fuzheng Jiedu Decoction on the Leukemia K562/A02 Multi-drug Resistance Cells and Its Mechanism / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To study the inhibitory effect of serum containing Fuzheng Jiedu decoction on leukemia multi-drug-resistance K562/A02 cells and its possible mechanism.</p><p><b>METHODS</b>The MTT method was used to detect the inhibitory rate of K562/AO2 cells treated with serum containing Fuzheng Jiedu decoction; the flow cytometry was used to detect the inhibitory effect of serum containing medicin on growth of K562/AO2 cells and P-gp expression; the Q-PCR was used to assay the BCL-2 mRNA expression; the Western blot was used to detect the BCL-2 protein expression.</p><p><b>RESULTS</b>MTT cytotoxic test showed serum containing Fuzheng Jiedu decoction could inhibit K562/A02 cell growth, and the inhibitory rate increased with the increase of drug concentration; the flow cytometry showed that the serum containing Fuzheng Jiedu decoction could promote K562/A02 cell apoptosis in a concentration-dependent manner. qPCR and Western blot showed that serum containing Fuzheng Jiedu decoction could down-regulate the protein expression of BCL-2. Fuzheng Jiedu decoction could reduce the protein expression of P-gp on the K562/A02 cell membrane.</p><p><b>CONCLUSION</b>serum containing Fuzheng Jiedu decoction can promote K562/A02 cell apoptosis, its mechanism of inducing apoptosis may be related with the inhibition of BCL-2 and P-gp protein expression.</p>

Screening for drug resistance related microRNAs in K562 and K562/A02 cell lines / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the relationship between microRNA and drug resistance in leukemia treatment by screening and identifying the microRNAs which differentially express in K562 cell line and its adriamycin resistant cells-K562/A02 cell line.</p><p><b>METHODS</b>The drug resistance potency of K562/A02 cells was evaluated by MTT assay. P-gp expression of K562 and K562/A02 cells were detected by flow cytometry (FCM). The differentially expressed microRNAs in K562 and K562/A02 cells were analyzed by microarray technique and Real Time RT-PCR.</p><p><b>RESULTS</b>The resistance to adriamycin (ADM) of K562/A02 cells was 180 fold greater than that of K562 cells. P-gp expression rate of K562 and K562/A02 cells was 0.2% and 86%, respectively. Twenty-two microRNAs expressed differentially in K562 and K562/A02 cells (P < 0.01). As compared to K562 cells, expressions of miR-221, miR-155 and miR-451 were up-regulated by more than two fold, while expression of miR-98, miR-181a, let-7f, let-7g, miR-424 and miR-563 down-regulated by more than two fold in K562/A02 cells. The results of real time RT-PCR were consistent with that of microarray. Of note, differential expressions of miR-451, miR-155, miR-221, let-7f and miR-424 were remarkable.</p><p><b>CONCLUSION</b>K562/A02 cells show a different microRNA expression profile as compared to its parental K562 cells, suggesting microRNAs including miR-221, miR-155, miR-451, let-7f and miR-424 may be involved in the mechanism of drug resistance in leukemia. These differentially expressed microRNAs provide potential novel targets for overcoming drug-resistance.</p>