Gm40600 suppressed SP 2/0 isograft tumor by reducing Blimp1 and Xbp1 proteins

Background Multiple myeloma (MM), characterized by cancerous proliferation of plasmablasts (PB) and plasma cells (PC), remains incurable in many patients. Differentially expressed molecules between MM PCs and healthy PCs have been explored in order to identify novel targets for treating MM. In the present study, we searched for novel MM therapeutic targets by comparing mRNA expression patterns between the Mus musculus myeloma plasmablast-like SP 2/0 cell line and LPS-induced PB/PC. Methods Gene expression profiles of LPS-induced PB/PC and SP 2/0 cells were determined using RNA-sequencing. A predicted gene (Gm40600) was found to be expressed at a low level in SP 2/0 cells. To study the role of Gm40600 in malignant PC, Gm40600 cDNA was cloned into a lentiviral vector (LV201) containing a puromycin selectable marker that was then transfected into SP 2/0 cells. Stable Gm40600-expressing SP 2/0 cells were selected using puromycin. The effect of Gm40600 on SP 2/0 cell proliferation, cell cycle/apoptosis, and tumor progression was assessed by cell counting kit-8 (CCK8), flow cytometry (FACS), and the SP 2/0 isograft mouse model, respectively. The effect of Gm40600 on mRNA and protein expression was evaluated by RNA-sequencing and western blotting, respectively. Results We found that SP 2/0 cells expressed lower level of Gm40600 mRNA as compared to LPS-induced PB/PC. Overexpression of Gm40600 significantly suppressed SP 2/0 cell proliferation and isograft tumor progression in an isograft mouse model by promoting apoptosis. In addition, Gm40600 overexpression suppressed transcription of the gene encoding Bcl2. Gm40600 overexpression also reduced the expression of PC-associated transcription factors Blimp1 and Xbp1, which promote transcription of the gene that encodes Bcl2. Conclusions Gm40600 reduced SP 2/0 cell proliferation and isograft tumor growth and progression by suppressing Blimp1 and Xbp1-mediated Bcl2 transcription to induce apoptosis. Thus, regulation of a human homolog of Gm40600, or associated factors, may be a potential therapeutic approach for treating MM. Electronic supplementary material The online version of this article (10.1186/s12885-019-5848-1) contains supplementary material, which is available to authorized users.

As cancerous cells, MM PC express MM-specific molecules that differ from healthy PCs [13][14][15]. To explore novel MM therapeutic targets, it is necessary to identify molecules that differ between healthy PC and MM PC. Because we found that Mus musculus myeloma PB-like SP 2/0 cells (MM PB/PC) expressed a significantly lower level of Gm40600 (a predicted gene) mRNA as compared to LPS-induced PB/PC (normal PB/PC), the effect of Gm40600 on SP 2/0 cell growth was tested.

Mice
Balb/c and CD19 cre mice have been previously described [16,17]. The Floxed Stch (Stch f/f ) mice in a B6 background were generated by Shanghai Biomodel Organism Science & Technology Development Co.,Ltd. (Shanghai, China). Stch f/f mice were crossed with CD19 cre mice to delete Stch in B cells. Gm40600 transgenic mice (cat no. TGB180522CEI02) were purchased from Cyagen Co., Ltd. (Guangzhou, China).

qPCR analysis
Total RNA was extracted from Vector-or Gm40600expressing SP 2/0 cells, and LPS-stimulated PB/PC with Trizol (Invitrogen Life Technologies). qPCR has been employed using a previous method [18] to quantify mouse Gm40600 gene expression. GAPDH mRNA expression is used to normalized relative mRNA expression that is then calculated relative to mRNA in SP 2/0 cells (set to 1).
Determination of IgM, IgG1, and IgG2a antibody levels by ELISA B cells from WT and Gm40600 transgenic mice were stimulated for 3 days in vitro with 10 μg/ml LPS. Antibody levels of in the supernatant were determined using mouse IgM, IgG1, and IgG2a ELISA kits (eBiosciences, Cat# 88-50,470-86, 88-50,140-22, and 88-50,420-88, respectively) as the instructions of the manufacturers.

Statistics
Cellular apoptosis, tumor volume, and tumor weight were compared between the vector alone group and the Gm40600 overexpressing group using the Student's t test. The change of cell proliferation at three different time points and the percentage of three different cell cycles phases (G1, S, and G2) were compared between the vector alone group and the Gm40600 overexpressing group using two-way ANOVA analysis. SEM was used as mean ± standard error of the mean. p < 0.05 value was considered to be statistically significant.

SP 2/0 cells express low levels of Gm40600 mRNA
Plasmablasts (PB) induced by LPS [20,22] and the Mus musculus myeloma PB-like SP 2/0 cells were used as normal PB/plasma cells (PC) and MM PB/PC, respectively. Both LPS-stimulated PB/PC and SP 2/0 cells expressed high levels of mRNA corresponding to the following PB/PC-associated transcription factors IRF4, Prdm1 (Blimp1), and Xbp1 (Table 1). Interestingly, as compared to LPS-stimulated PB/PC, SP 2/0 cells expressed a significantly lower level of Gm40600 mRNA (Table 1). Thus, reduction of Gm40600 may promote malignant growth of SP 2/0 cells.

SP 2/0 cell proliferation was suppressed by Gm40600
To explore the effect of Gm40600 in malignant growth of SP 2/0 cells, an LV201 construct expressing Gm40600 were transfected into SP 2/0 cells. Stable transfectants expressing Gm40600 or Vector were selected by puromycin. qPCR assay demonstrated that Gm40600 mRNA was significantly increased in stably transfected SP 2/0 cells and lower than that in LPS-induced PB/PC (Additional file 1: Figure S1). Critically, CCK8 assay showed that cell number is time-dependently lower in Gm40600-expressing SP 2/0 cells than that in vectorexpressing SP 2/0 cells (Fig. 1a). Furthermore, cell cycle and apoptosis analysis suggested that Gm40600 overexpression did not affect the cell cycle but it did promote cell apoptosis (Fig. 1b-d). Collectively, these results demonstrated that Gm40600 significantly suppressed proliferation of SP 2/0 cells by promoting apoptosis.
To explore the mechanism by which Gm40600 regulates transcription, the location of Gm40600 in SP 2/0 cells was examined. SP 2/0 cells expressing either Gm40600-EGFP or EGFP alone were imaged on a GE IN Cell Analyzer 2000. The results demonstrated that EGFP alone was distributed throughout the cells, whereas Gm40600-EGFP was mainly localized to the cytoplasm (Fig. 3a). These results suggest that Gm40600 is not a transcription factor.

Gm40600 overexpression suppressed antibody production in LPS-stimulated primary B cells
To study the role of Gm40600 in normal PC, we found that Stch (a gene coding the heat shock protein family A [Hsp70] member 13) was mainly expressed in PC and a B cell-specific knock-out of Stch did not affect B-cell mature and activation but reduced PC production (data not shown). In this work, Stch deficiency reduced LPS-induced prdm1 and xbp1 mRNA by 50% (Table 3). Accordingly, LPS-stimulated Stch −/− B expressed almost no Gm40600 (Table 3). Collectively, these data suggest that Gm40600 expression may positively correlate with Prdm1 and Xbp1 expression and involved in nonmalignant PC generation/maintenance. To further explore the function of Gm40600 in antibody production, Gm40600 transgenic mice were developed. LPS can effectively induce PC production and antibody secretion [20]. Thus, LPS was used to induce antibody production in B cells from wild-type (WT) and Gm40600 transgenic mice. As expected, Gm40600 overexpression suppressed antibody production in LPSstimulated primary B cells (Fig. 4).

Discussion
Untill now, the expression pattern of Gm40600 remains unclear and its function is uncharacterized. RNAsequencing demonstrated that LPS-induced PB/PC expressed a high level of Gm40600 mRNA, whereas Fig. 1 Gm40600 overexpression suppressed proliferation and promoted apoptosis of SP 2/0 cells. a Gm40600 overexpression suppressed SP 2/0 cell proliferation. Gm40600-or Vector-expressing SP 2/0 cells were cultured in vitro for 0, 1, and 3 days. CCK8 assay was used to analyze to cell proliferation. b-d Gm40600 overexpression affected SP 2/0 cell apoptosis but not cell cycle. Gm40600-or Vector-expressing SP 2/0 cells were cultured in vitro for 2 days. Cells were then stained and analyzed using propidium solution (PI) and flow cytometry (FACS), respectively. FACS profile, cell cycle, and apoptotic cell percentages are shown in (b, c, and d), respectively. d Two-tailed Student's t test and a, and b two-way ANOVA followed by Bonferroni post-tests to compare each column to control column. Error bars, SEM. **p < 0.01, ****p < 0.0001 cancerous SP 2/0 cells expressed a low level of Gm40600 mRNA (Table 1). In addition, Gm40600 was identified as a suppressive molecule that suppresses cancerous SP 2/0 cell proliferation (Fig. 1a) and isograft tumor progression (Fig. 2). With a high incidence of survival because of usage of these drugs such as bortezomib, thalidomide and lenalidomide [27,28], MM is still incurable and novel therapeutic strategies are needed [28]. To explore potential therapies, more knowledge of MM biology is needed [28,29]. This work demonstrated that cancerous SP 2/0 cells expressed very low levels of Gm40600 (Table 1), whereas Gm40600   1 and 2). In addition, Gm40600 mRNA was significantly lower in stably transfected SP 2/0 cells than that in LPS-induced PB/PC (Additional file 1: Figure S1). These results suggest that overexpression of Gm40600 in SP 2/0 cells is lower than its physiological expression in LPS-induced PB/PC. This supports that the downregulation of Gm40600 in SP 2/0 cells is a necessary part of tumor development. Thus, it is worth to identify a human homolog for Gm40600 and explore the role of a human homolog of Gm40600 in MM development.
The survival of malignant PC [30], including MM cells [31,32], was maintained by anti-apoptosis molecules. This inspired researchers to target these anti-apoptotic proteins as a strategy for treating MM [33]. Apoptosisinduced drugs such as Bortezomib have been approved to treat MM [34]. Consistent with these studies, we demonstrated here that Gm40600 overexpression induced SP 2/0 cell apoptosis (Fig. 1b-d).
Finally, we found that Gm40600 expression is positively related to Prdm1 and Xbp1 expression involved in nonmalignant PC generation/maintenance (Table 3). However, Gm40600 overexpression suppressed antibody production in LPS-stimulated primary B cells (Fig. 4). Collectively, these data suggest that Gm40600 is a negative regulator of antibody production.
There are still some unsolved questions: 1, the mechanisms by which Gm40600 reduces Blimp1; 2, the physiological function of Gm40600 in Prdm1 and Xbp1 expression, and nonmalignant PC generation/maintenance; 3, whether there are human orthologs of the Gm40600 gene; 4, the pathogenic role of the human ortholog of Gm40600 gene in MM; 5, whether regulation of the Gm40600 human ortholog can be used to treat MM. Researching these questions will be important for understanding the role of Gm40600 and its human orthologs in MM.

Conclusions
Compared to LPS-induced PC, cancerous SP 2/0 cells expressed a low level of Gm40600 mRNA. Overexpression of Gm40600 suppressed SP 2/0 cell proliferation and isograft tumor growth by reducing Blimp1 and Xbp1-mediated transcription of the anti-apoptosis molecule Bcl2. Thus, it is necessary to explore the role of a human homolog of Gm40600 in MM. This will guarantee that regulation of a human homolog of Gm40600, or associated factors, may represent a novel therapeutic strategy for treating MM.

Additional file
Additional file 1 : Figure S1. Gm40600 mRNA expression was significantly lower in stable Gm40600-expressing SP 2/0 cells than that in LPS-induced plasmablasts/plasma cells (PB/PC). B cells from the splenocytes of three 8-9-week-old Balb/c mice per group were sorted by B220 microbeads, and stimulated for 3 days in vitro by 10 μg/ml LPS. The stable Vector-or Gm40600-expressing SP 2/0 cells were thawed, passaged for 3 times and then cultured for 3 days in fresh medium. Gm40600 mRNA expression was determined by qPCR in Vector-expressing SP 2/0 cells (Vector-SP2/0), Gm40600-expressing SP 2/0 cells (Gm40600-SP2/0) and LPSstimulated PB/PC (LPS-PB/PC) were determined. Relative mRNA levels are normalized to GAPDH mRNA expression and calculated relative to the mRNA expression in SP 2/0 cells, set to 1. Figure S2. Gm40600 overexpression reduced Blimp1 and Xbp-1 protein in SP 2/0 cells. Vector-or Gm40600-expressing SP 2/0 cells were thawed, passaged for 3 times and then cultured for 3 days in fresh medium. The cells were then collected and subjected to immunoblot analysis with monoclonal anti-mouse antibodies for Xbp1, Blimp1, and β-tubulin. Results represent three independent experiments. Figure S3. Gm40600 overexpression suppressed the Bcl2 promoter activation in SP 2/0 cells. The luciferase reporter vector pEZX-PG04.1/Bcl2 promoter (− 1323~+ 160 bp) and renilla luciferase reporter vector pRLSV-40 vector were co-transduced into stable Gm40600or vector-expressing SP 2/0 cells. The cells were cultured for 3 days. Dual luciferase reporter gene expression was analyzed, and the results are shown as the ratio of firefly to renilla luciferase activity. The data represent three independent experiments. Error bars, SEM. Two tailed Student's t-test, **P < 0.01. (PDF 203 kb)  Fig. 4 Gm40600 overexpression suppressed antibody production. 10 μg/ml LPS was used to stimulate B cells from the splenocytes of 7-to 9week-old WT and Gm40600 transgenic mice (three mice per group, total three independent experiments). On 3 days, IgM, IgG1, and IgG2b levels in cell culture supernatant was analyzed using ELISA assay. Error bars, SEM. Two tailed Student's t-test, *P < 0.05