Background
Each year, deaths due to multiple myeloma (MM) with malignant plasma cells (PC) [
1] is approximately 1% of all cancer deaths [
2,
3]. This is mainly because of the following two reasons: (1) MM is still incurable in many patients [
4,
5]; (2) some patients are refractory to current conventional drugs [
6,
7]. Thus, it is urgent to find novel therapeutic way to treat MM [
6].
Several reports have shown that MM PC share many common characteristics with healthy PC [
2,
3,
8]. Normally, the differentiation of activated B cells into PC is strictly controlled by several genes, including B cell-associated Pax5, germinal center (GC) B cell-associated Bcl6, and PC-specific genes such as IRF4, Prdm1 (Blimp1), and Xbp1 [
9,
10]. Bcl2 also promotes B cell differentiation into PC, as Bcl2 with anti-apoptotic activity supports GC B cells and PC survival [
11,
12].
As cancerous cells, MM PC express MM-specific molecules that differ from healthy PCs [
13‐
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.
Methods
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).
RNA-sequencing
B220
+ B cells were sorted from splenocytes of 7- to 9-week female Balb/c, Stch
f/f, and CD19
creStch
f/f mice (3 mice per group) using B220 microbeads (Cat No. 130–049-501, Miltenyi Biotec, Germany), B220
+ B cells were stimulated with 10 μg/ml LPS (L2630, Sigma, St Louis, MO) for 3 days in vitro as previously described [
18]. SP 2/0 cells (ATCC® CRL-1581, Rockville, MD, USA) were thawed, passaged three times, and then cultured for 2 days in fresh medium. RNeasy Mini Kit (Qiagen, Venlo, Netherlands) was used to isolate and purify total RNA from cells. NanoDrop®ND-1000 spectrophotometer and Agilent 2100 Bioanalyzer and RNA 6000 NanoChips (Agilent, Palo Alto, CA, USA) were used to determine RNA concentration and quality, respectively. TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold (Illumina) was used to prepare Libraries. Transcripts were analyzed by RNA-sequencing (Genewiz Corp., Suzhou, China) using a standard method [
18].
qPCR analysis
Total RNA was extracted from Vector- or Gm40600-expressing 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).
Effect of Gm40600 on SP 2/0 growth
Gm40600 cDNA (accession: XM_011243239) was synthesized by General Biosystems Corp. (Anhui, China) and subcloned into LV201 (Fugene Corp., Guangzhou, China), a lentiviral vector with a puromycin selectable marker. Gm40600-expressing LV201 or control LV201 (empty vector) were transfected into SP 2/0 cells using a previously described method [
18,
19]. Puromycin (10 μg/ml; Sigma) was used to select stable transfectants. Counting kit-8 (CCK8), a propidium iodide (PI)/FACS, and the SP 2/0 isograft mouse model were previously described [
18,
20,
21] and used to assess the effect of Gm40600 on SP 2/0 cell proliferation, cell cycle, and isograft tumor progression, respectively.
Gm40600 localization and protein expression
Gm40600 was cloned into EGFP-expressing LV122 (Fugene Corp., Guangzhou, China) to express a Gm40600-EGFP fusion protein. Gm40600-EGFP-expressing LV122 was transfected into SP 2/0 cells using a previously described method [
18]. Gm40600-EGFP protein localization was analyzed by immunofluorescence and confocal microscopy according to a previously described method [
18]. The effect of Gm40600 on Bcl6, IRF4, Blimp1, Xbp-1, Trp53, and Bcl2 was determined by western blotting using a previously described method [
18]. The western blots were probed with rabbit against mouse Bcl6 (sc-858, Santa Cruz Biotech), IRF4 (ab104803), Blimp1 (sc-25,380, Santa Cruz Biotech), Xbp-1 (ab37152, Abcam), Trp53 (#2524, Cell Signaling Tech), Bcl2 (ab59348, Abcam) and β-tubulin (KM9003T, SunGene Biotech) antibodies.
Lv81/Blimp1 (0.5 μg, cat# EX-Mm24401-Lv81, Fugene Corp., Guangzhou, China), pEZX-PG04.1/Bcl2 promoter (0.5 μg, cat# MPRM19639-PG04, Fugene Corp., Guangzhou, China), and pRLSV-40 vector (0.05 μg, cat# E2231, Promega Corp.) were co-transduced into 4 × 10
5 293 T cells according to our previous study [
18]. In some experiments, firefly luciferase reporter plasmid pEZX-PG04.1/Bcl2 promoter and renilla luciferase reporter vector pRLSV-40 vector were transduced into stable Gm40600- or vector-expressing SP 2/0 cells. On day 3, 1420 Multilabel Counter (1420 Victor 3, PerkinElmer Corp.) was used to determine the activity of firefly and renilla luciferase.
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.
Discussion
Untill now, the expression pattern of Gm40600 remains unclear and its function is uncharacterized. RNA-sequencing demonstrated that LPS-induced PB/PC expressed a high level of Gm40600 mRNA, whereas 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 overexpression suppressed cancerous SP 2/0 cell growth (Figs.
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]. Apoptosis-induced 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).
An important regulator in cell apoptosis is Bcl-2 [
35,
36]. Studies have shown that several human tumors aberrantly overexpress Bcl-2, including MM [
37,
38]. However, the mechanisms underlying aberrant Bcl-2 expression remain largely unknown [
39]. Our data demonstrated that Gm40600 reduced both Bcl2 mRNA and protein in SP 2/0 cells (Table
2, Fig.
3b).
Xbp1 can effectively induce Bcl2 transcription by directly binding to CTG-CRE sites in the Bcl2 gene promoter or indirectly by ESR1 [
25,
26,
40]. Our work demonstrated that Gm40600 reduced Bcl2 mRNA (Table
2) by suppressing Bcl2 promoter activation (Additional file
1: Figure S3), and Xbp1 protein (Fig.
3b, Additional file
1: Figure S2) in SP 2/0 cells. This suggests that Gm40600 suppresses Bcl2 transcription by reducing Xbp1 protein levels. Previous studies have shown that Blimp-1 induces expression of Xbp1 [
41‐
43], which promotes antibody production [
44,
45]. Our data showed that both Blimp1 and Xbp1 protein levels were reduced in SP 2/0 cells overexpressing Gm40600 (Fig.
3b, Additional file
1: Figure S2). Collectively, these results suggest that Gm40600 induced apoptosis by reducing Blimp1 and Xbp1-mediated Bcl2 transcription.
Blimp1 promotes the survival of PC in healthy donors and MM patients, whereas its deficiency causes PC apoptosis [
31,
46]. In accordance with these studies, our data demonstrated that overexpression of Gm40600 reduced both Blimp1 expression (Fig.
3b) and promoted apoptosis (Fig.
1b-d). Thus, Gm40600 overexpression promoted SP 2/0 cell apoptosis by reducing Blimp1 expression.
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.
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