Expression pattern of MRPS18 family genes in malignantly transformed b-cells
Summary. Aim: To compare expression patterns of proteins of a family of mitochondrial ribosomal protein S18 (MRPS18) in tumor cell lines of the B-cell origin. Materials and Methods: The study has been performed on different subsets of tonsil B-cells and tumor cell lines of the B-cell origin using quantitative polymerase chain reaction analysis, western blot analysis, immunohistochemistry, bioinformatic analysis of the publicly available data bases on expression. Results: We have found that genes of the MRPS18 family (1–3) show different expression patterns in tumor cell lines of the B-cell origin. The highest levels of expression were shown for MRPS18-3, the lowest — for MRPS18-1. MRPS18-2 was expressed at the highest levels in germinal center cells, Burkitt lymphoma and Hodgkin lymphoma cell lines. At the protein levels, MRPS18-2 showed the highest expression in Burkitt lymphoma and B-cell precursor acute lymphoblastic leukemia cell lines. In lymphoblastoid cell lines, and in germinal center B-cells MRPS18-2 levels were somewhat lower, but higher than in memory and plasma B-cells. Conclusions: The differential expression pattern of the MRPS18 family proteins suggests that they play various roles in cellular processes.
Submitted: November 15, 2020.
*Correspondence: E-mail: Kashuba@nas.gov.ua;
Abbreviations used: BCP-ALL — B-cell precursor acute lymphoblastic leukemia; BL — Burkitt’s lymphoma; EBV — Epstein — Barr virus; GC — germinal center; HL — Hodgkin lymphoma; LCL — lymphoblastoid cell line; MRPS18 — mitochondrial ribosomal protein S18; MZ — mantle zone of GC; qPCR — quantitative polymerase chain reaction; XLP — X-linked lymphoproliferative disease.
A family of mitochondrial ribosomal proteins of the small subunit (MRPS18) consists of three members, 1–3. They are homologues and have one bacterial ancestor . The properties of these proteins are largely unknown, though. Earlier, we have demonstrated that the MRPS18-2 protein plays an important role in cell immortalization and transformation [2–4]. Moreover, expression of MRPS18-2 was elevated in various types of malignancies, such as endometrial , prostate , liver  and breast  cancers. Importantly, the MRPS18-2 protein is involved in the control of cell stemness . Other proteins of this family also demonstrate properties beyond the mitochondrial protein function, for example, MRPS18-3 is overexpressed in breast cancer . Of note, expression of the MRPS18-1 gene in fibroblasts with the mutated MT-ND1 gene could overcome negative consequences of mutation  that once again demonstrates the important function of MRPS18 family genes.
In the present work, we aimed to compare expression pattern of MRPS18 members at mRNA and protein levels in tonsil and in malignantly transformed B-cells.
MATERIALS AND METHODS
Cell lines. In the present work, the following cell lines of the B-cell origin were used: NALM6 and REH, produced from B-cell precursor acute lymphoblastic leukemia (BCP-ALL); Burkitt lymphoma (BL) — RAMOS and BJAB (Epstein — Barr virus (EBV) negative, DAUDI — EBV positive of Latency I, RAJI — EBV positive of Latency III; Hodgkin lymphoma (HL) — L-1236, KM-H2, L-428; and EBV transformed lymphoblastoid lines T5-1 and MP1, and also EBV transformed lymphoblastoid lines from patients with X-linked lymphoproliferative disease (XLP) — 8003 and IARC-739 kindly provided by Prof. K.E. Nichols (USA). Lymphoblastoid cell lines (LCL) were described earlier [12, 13]. All other cells are from the Bank of Cell Lines from Human and Animal Tissues of RE Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology of NASU, Kyiv, Ukraine. Cells were grown in IMDM medium, containing 10% fetal bovine serum, 2 mM L-glutamine, and appropriate antibiotics. Cell lines used in this study were screened for mycoplasma infection and conﬁrmed to be negative.
Isolation of tonsil B-cells. Tonsils were obtained from patients undergoing tonsillectomy. The usage of tonsil tissue and the archived biopsy specimens of HL was approved by the Institutional Review Board and Research Ethics Committee of RE Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology of NASU according to the Declaration of Helsinki. All experimental work was performed, according to the approved protocols. Isolation of the B-cell subsets was described earlier in detail .
RNA isolation, cDNA synthesis and quantitative polymerase chain reaction (qPCR). The total RNA was isolated, using the RNeasy Mini Kit (Qiagen Inc., Germany), according to the manufacturer’s instructions. The cDNAs were synthesized, using 2 μg of total RNA, M-MLV Reverse Transcriptase and RNAse inhibitor (Invitrogen, USA), according to the manufacturer’s protocol. q-PCR was performed, using 2 μg cDNA and the SYBR Green Master Mix (Thermo Fisher Scientific Inc., USA), on the PCR System 7500 (Applied Biosystem, USA). Primers were following: for MRPS18-1(NM_016067) forward — 5’-CAGGTATCCAGCAATGAGGACC-3’, reverse 5’-GCATCCAGTAAATGGAGAAACAAAC-3’; for MRPS18-2 (NM_014046) — forward 5’-CACAGCGG ACTCGGAAGACATG-3’, reverse 5’-GCGCAGACAAATTGCTCCAAGAG-3’; for MRPS18-3 (NM_018135) — forward 5’-CATCTGCCGTTGGAACCTTGAAG-3’, reverse 5’-CTTGCGGTGTT CTTCCTGGCAT-3’. As an internal control for standardization, a gene encoding TATA-binding protein (TBP, NM_003194) was used: forward primer — 5’-TTTCTTGCCAGTCTGGAC-3’, reverse 5’-CACGAACCACGGCACTGATT-3’.
Western blotting. Total cell lysates were prepared, using the NP40 lysis buffer (1% NP40, 150 mM NaCl, 50 mM Tris, pH 8) with a protease inhibitor cocktail (Roche AB, Stockholm, Sweden) and bovine serum albumin, BSA (0.5% w/v) as a nonspecific competitor. After SDS–PAGE, proteins were transferred to nitrocellulose and probed with rabbit antibodies against MRPS18-2 (Life Technologies, Carlsbad, CA, USA), and actin (Sigma-Aldrich). Secondary antibodies (anti-mouse IgG HRP conjugated) were from GE Healthcare Bio-Sciences AB, Uppsala, Sweden.
Immunohistochemistry. Expression of the MRPS18-1-3 proteins was determined on the paraffin-embedded tissue sections. Rabbit polyclonal anti-MRPS18-1, -2 and -3 (Proteintech Group Inc., Chicago, IL, USA) 1:100 antibodies were used. This procedure is described in detail earlier .
Bioinformatic data analysis. To analyze expression of genes at the mRNA level, a publicly available data base Oncomine was used. This data base contains published data that have been collected, standardized, annotated and analyzed by Compendia Bioscience (www.oncomine.com, November 2020, Thermo Fisher Scientific, Ann-Arbor, MI, USA).
Statistical analysis. GraphPad Prism software (version 7, GraphPad Software, La Jolla, CA, USA) was used to determine the means of the MRPS18 gene expression, and for MRPS18-2 in a western blot analysis, as a normalized ratio of protein to actin signals, in the studied cell lines.
RESULTS AND DISCUSSION
Relative expression of the MRPS18 family genes, assessed by qPCR, was normalized to such in a total population of tonsil B-cells (before fractionation of B-cells). We observed the differential expression of all three genes of the MRPS18 family in various cells. The lowest expression was detected for the MRPS18-1 gene, the highest — for MRPS18-3. Moreover, an expression pattern differs as well between these three proteins. Thus, MRPS18-1 was expressed at low levels in all studied cell line, and in HL and LCL expression was decreased further (Fig. 1, a).
MRPS18-2 gene showed the highest expression in the established cell lines, especially in BL cells, compared to any subset of the peripheral blood B-cells (Fig. 1, c).
MRPS18-3 gene showed the highest expression, compared with MRPS18-1 and MRPS18-2. Of note, expression was 3–4 folds elevated in all cells and cell lines studied, with exception of the total tonsil and naїve B-cells (Fig. 1, e).
To corroborate our experimental data, a bioinformatic analysis was performed, using the Oncomine portal. Importantly, similar to our results, differential expression was found for all three genes (Fig. 1, b, d, e) . Indeed, the lowest expression in B-cells and in tumors of the B-cell origin, such as BL, HL, DLBCL, etc., was found for the MRPS18-1 gene (Fig. 1, b), and the highest — for MRPS18-2 (Fig. 1, f). Noteworthy, mRNA level of MRPS18-2 was quite high in BLs, DLBCLs and a subset of HLs, but the signal was lower, compared to such of MRPS18-3 in the same samples (Fig. 1, d and f).
Fig. 1. An expression pattern of the MRPS18 family genes at mRNA levels. Expression of MRPS18-1 in the studied cells, assessed by qPCR (a) and retrieved from Oncomine  (b). Similarly, expression of MRPS18-2 (c) and (d), and of MRPS18-3 (e) and (f) was studied. Ct values were determined for the internal control (TBP) and for the test genes at the same threshold level in the exponential phase of the PCR curves. Relative quantification (comparative Ct (ΔΔCt) method) was used for comparison of gene expression with the internal control. Two (or three) reactions, each in triplicate, were run; the standard deviation was calculated. The Kruskal — Wallis test for non-parametric values in groups was applied for each sample. P values were calculated as p = 0.0003, p < 0.0001, p < 0.0001 for MRPS18-1, -2 and -3, correspondingly
Next, we wanted to assess expression pattern of MRPS18-2 at the protein level in different B-cells and tumor cell lines of B-cell origin, because this gene showed the most dramatic changes in expression levels. To do so, the western blot analysis was performed. LCLs, established form XPL patients, were omitted this time, and cell lysate of BCP-ALL cell line REH was loaded in duplicates (Fig. 2, a). After densitometry was performed, the ratio of MRPS18-2 to actin signals was calculated, and the data were analyzed, using the Kruskal–Wallis test for non-parametric values in groups (Fig. 2, b). Indeed, at the protein levels, MRPS18-2 showed the highest expression in BL and BCP-ALL cell lines. In LCLs, and in germinal center (GC) B-cells MRPS18-2 levels were somewhat lower, but higher, than in memory and plasma B-cells.
Fig. 2. Expression pattern of MRPS18-2 at the protein level in different B-cells and tumor cell lines of B-cell origin. The western blot analysis (a) and the calculated ratio between MRPS18-2 and actin signals (b)
Bearing in mind that any cell line possesses only few characteristics of tumor cells in situ, due to selective forces while growing in vitro, we next examined expression patterns of the MRPS18 family proteins in tonsil and few histological samples of HL. We have to mention that, of course, antibodies against abovementioned proteins have various affinity. From other hand, the MRPS18 proteins might have different stability. Nevertheless, we found that MRPS18-2 protein showed a signal of the highest intensity in follicular B-cells of GC, and also in HL cells (Fig. 3, c and g), compared to MRPS18-1 (Fig. 3, b and f) and MRPS18-3 (Fig. 3, d and h).
Fig. 3. Expression levels of the MRPS18 family proteins in tonsil (a–d) and HL (e–h) primary tissues. MRPS18-1 and MRPS18-3 expresses mainly in follicular zone of GC (b and c), but not in the mantle zone (indicated by mantle zone of GC (MZ)), while MRPS18-2 signal is seen also in MZ cells (d). Reed-Steinberg cells are present in HL tissue sample (e, indicated with arrows). The most intensive signal is registered for MRPS18-2 (g), and not for MRPS18-1 (f) and MRPS18-3 (h)
The obtained results are in concordance with our findings, reported previously: the MRPS18-2 protein levels are high in tumor cells, for example, in endometrial , prostate , liver  and breast  tumors. Obviously, the MRPS18 proteins have different biochemical properties, and their functions, including stability and a protein degradation rate should be further investigated.
We have found that genes of the MRPS18 family (1–3) show different expression patterns in tumor cell lines of the B-cell origin. The highest levels of expression were shown for MRPS18-3, the lowest — for MRPS18-1. MRPS18-2 was expressed at the highest levels in GC cells, BL and HL cell lines. The differential expression pattern of the MRPS18 family proteins suggests that they play various roles in cellular processes. Biochemical properties and a functional role of the MRPS18 proteins in carcinogenesis should be further elucidated in the future studies.
This work was supported by the Emil and Wera Cornells Foundation (094-2020) and by the Academy of Science of Ukraine (grant № 0119U103905).
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