miR-100-5p is upregulated in multiple myeloma and involves in the pathogenesis of multiple myeloma through targeting MTMR3

ABSTRACT Objectives: MicroRNA (miRNA) is a kind of highly conserved single-stranded small endogenous non-coding RNA associated with multiple diseases, particularly cancer. The miRNAs expression profile in multiple myeloma (MM) has been barely elucidated. Methods: The miRNAs expression profiles in bone marrow plasma cells of 5 MM individuals and 5 iron-deficiency anemia volunteers were analyzed using RNA-sequencing. Quantitative polymerase chain reaction (QPCR) was performed to validate the expression of selected miR-100-5p. The biological function of selected miRNA was predicated by bioinformatics analysis. Finally, the function of miR-100-5p and its target on MM cells were evaluated. Results: MiRNA-sequencing showed that miR-100-5p was obviously upregulated in MM patients, which was further validated in an expanded cohort. Receiver operating characteristic curve analysis characterized miR-100-5p as a valuable biomarker of MM. Bioinformatics analysis predicted that miR-100-5p is targeted to CLDN11, ICMT, MTMR3, RASGRP3, and SMARCA5, and their low expression are associated with poor prognosis of MM patients. Kyoto encyclopedia of genes and genomes analysis suggested that the major interacting proteins of these five targets are mainly enriched in inositol phosphate metabolism and phosphatidylinositol signaling system pathway. In vitro study showed that miR-100-5p inhibition promoted the expression of these targets, especially MTMR3. In addition, miR-100-5p inhibition declined living number and metastasis, whereas promoted apoptosis of RPMI 8226 and U266 MM cells. The function of miR-100-5p inhibition was weakened by MTMR3 inhibition. Conclusion: These results indicates that miR-100-5p is a promising biomarker for MM, and that it may involve in the pathogenesis of MM by targeting MTMR3.


Introduction
Multiple myeloma (MM) is the most prevalent type of malignant plasma cell diseases, characterized by abnormal proliferation of bone marrow plasma cells and overproduction of monoclonal immunoglobulin or light chain, accounting for 1% of all malignancies and 10% of hematological malignancies [1,2].Its clinical manifestations are bone pain and fracture, anemia, renal insufficiency, hypercalcemia, and infection, with significant heterogeneity [2].Despite the development of novel drugs, such as immunomodulatory agents (lenalidomide and pomalidomide) and proteasome inhibitors (bortezomib, ixazomib, and carfilzomib), MM is still incurable and often occurs relapse [3,4].Therefore, it is urgent to understand the molecular mechanism of MM, uncovering new therapeutic targets to develop novel treatment strategy and improve the prognosis of MM patients.MicroRNA (miRNA) is a kind of highly conserved single-stranded small endogenous non-coding RNA and widely exists in various eukaryotes, showing an important regulatory effect in many biological processes, such as proliferation, differentiation, apoptosis, and signal transduction of somatic cells [5,6].Accumulating studies have confirmed that miRNA is involved in the progression of various tumors by regulating proliferation, apoptosis, differentiation, and metastasis of tumor cells [7][8][9].Evidences has demonstrated that heterogeneously expressed miRNAs show a crucial function in MM pathology, including tumor initiation, progression, metastasis, prognosis, and drug response [10].MiRNAs are involved in regulating the interaction between myeloma cells and bone marrow microenvironment in MM. miRNA can affect the immune and metabolic state of bone marrow microenvironment, and microenvironment changes also affect the nutritional environment of myeloma cells, indicating miRNAs as expected targets and prognostic biomarkers for MM therapy [11].As reported by Papadimitriou et al., miR-181a is upregulated in MM patients, strongly associated with inferior disease outcome, demonstrating miR-181a as a personalized prognostic biomarker and treatment target [12].
Herein, we first evaluated the miRNAs expression profiles in bone marrow plasma cells of 5 MM individuals (MM group) and 5 iron-deficiency anemia volunteers (Control group), founding that miR-100-5p was significantly overexpressed in MM samples, which was validated using quantitative polymerase chain reaction (qPCR).Moreover, we predicted the biological function of miR-100-5p by bioinformatics analysis, and investigate the potential function of miR-100-5p in the pathogenesis of MM.

Study population
A total of 21 patients with MM admitted to Gansu Provincial People's Hospital from July 2021 to June 2022 were selected, and another 30 patients with iron deficiency anemia admitted at the same time were set as control group.Patients were diagnosed as MM according to the criteria of International Myeloma Working Group 2014 (IMWG) [13].MM patients with the presence of other malignancies, complicated with other autoimmune diseases and hematological malignancies, during pregnancy and lactation, or with mental disorders and inability to cooperate with treatment were excluded.The baseline characteristics of patients were collected, including age, gender, leukocytes, hemoglobin, platelets, albumin, globulin, β2 microglobulin, blood calcium, lactate dehydrogenase, diagnosis stage (DS, ISS, and R-ISS), ECOG performance score, proportion of plasma cells, and extramedullary lesions.The details were shown in Table 1.All individuals received 4 mL of bone marrow blood isolated plasma cells for RNA sequencing.The research was approved by the Ethics Committee of Gansu Provincial People's Hospital (Approval number: 2022-234).All patients voluntarily participated in the trial and signed informed consent.

RNA sequencing (RNA-seq)
Total RNA was extracted from bone marrow plasma cells using TRIzol reagent (Invitrogen, Cat.NO:15596026) according to the methods reported by Chomczynski et al. [14].Thereafter, DNA digestion was carried out using DNaseI.RNA quality was determined by examining A260/A280 using NanodropTM OneCspectrophotometer (Thermo Fisher Scientific Inc), and its integrity was confirmed by 1.5% agarose gel electrophoresis.The collected qualified RNAs were finally quantified by Qubit3.0 using QubitTM RNA Broad Range Assay kit (Life Technologies, Cat. NO: Q10210). 3 μl of extracted RNA in each sample was utilized for miRNA library preparation using small RNA Library Prep Kit for Illumina following the manufacturer's instruction.The eluted cDNA library was separated by 6% PAGE gel, and ∼160 bp bands were isolated, purified, and quantified by Qubit3.0,subsequently sequenced using Hiseq X-10 sequencer (Illumina) with PE150 model.

miRNA-Seq data analysis
To obtain clean reads, low-quality reads were filtered using fastx_toolkit software (version: 0.0.13.2), and adaptor sequences were trimmed using cutadapt software (version: 1.15).Clean Reads were then treated with in-house scripts to eliminate duplication bias introduced in library preparation and sequencing.In brief, clean reads were clustered according to the UMI sequences, in which reads with the same UMI sequence were grouped into the same cluster.Reads in the same cluster were compared to each other by pairwise alignment, and then reads with 100% sequence identity were extracted to a new subcluster.Thereafter, multiple sequence alignment was performed to get one consensus sequence for each sub-cluster to eliminate any errors and biases introduced by PCR amplification or sequencing.The deduplicated consensus sequences were mapped to the human reference genome (GRCh37/HG19), using bowtie (version: 1.1.2) with default parameters.The package of mirdeep2 (version:2.0.0.8) was used for mapping the reads to the known primary-miRNA in the database of miRBase and predicting the novel miRNA.The miRNA differential expressed between groups was identified using the edgeR package (version: 3.12.1).A cutoff of p-value < 0.05 and | Log2Fold-change| >1 were used to evaluate the statistical significance of miRNA expression differences.

Cell counting kit-8 assay
Cell counting kit-8 (CCK-8) assay was conducted to detect the living number of RPMI 8226 and U266 MM cells.3 × 10 5 harvested RPMI 8226 and U266 cells were seeded in 96-well plates (100 μL of medium per well), and maintained at 37°C with 5% CO 2 for 24, 48, and 72 h.Thereafter, the cells were cultured for 4 h with 10 μL of CCK-8 solution (Solarbio, Beijing, China) and the optical density was measured at 450 nm using an AMR-100 apparatus (Allsheng).

Flow cytometry
The apoptosis rate of MM cells was examined using flow cytometry.The Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis assay kit (BD Biosciences, #556547) was utilized.1 × 10 6 cells were centrifuged at 400× g, 4 °C for 5 min, followed by being resuspended in 200 ′ μL of phosphatebuffered saline (PBS).Thereafter, the cells were cultured with 200 µL of binding buffer, stained for 30 min with 10 μL of Annexin V-FITC and 10 μl of PI in the dark.Then, 300 µL of binding buffer was added and the cells were subjected to flow cytometry using a Novocyte apparatus (ACEA Biosciences, Inc., U.S.A.).

Results
MiR-100-5p is identified as a biomarker of MM A total of 10 RNA-seq libraries were contrasted using bone marrow plasma cell samples of 5 MM patients and 5 control individuals.The raw reads and bases, valid reads and bases, Q20, Q30, and GC percentage were collected from each library, the corresponding details were shown in Table 2, showing that Q20 > 98%, Q30 > 94%, and GC content ranged from 45% to 49%.These indicated the high quality of the RNAseq data and guaranteed the reliability of follow-up analysis.MiRNA-seq data analysis showed that there were 44 significantly differentially expressed miRNAs between MM patients and control individuals, of which 12 miRNAs were downregulated and 32 miRNAs were upregulated in MM patients, especially miR-100-5p (Figure 1A-C).The detailed information of all the expressed miRNAs were shown in Supplementary 1.Thus, we speculated that miR-100-5p may be involved in the pathogenesis of MM.Furthermore, validation experiment revealed that miR-100-5p were significantly highly expressed in bone marrow of MM patients compared to those of control individuals (p < 0.001) (Figure 1D), which was consistent with the RNA-seq analysis.Moreover, the clinical utility of miR-100-5p was evaluated using ROC curve analysis.The results showed that the area under the curve of miR-100-5p reached 0.983 (95% confidence interval, 0.9484-1.018;p < 0.0001) (Figure 1E), thereby indicating that miR-100-5p may represent valuable biomarker of MM.

Targets predication of miR-100-5p in MM
Then, we predicated the targets of miR-100-5p using miRanda, miRWalk, and PITA databases, showing that 10 targets can be found in all of these three databases including ICMT, RAP1B, RCBTB1, RASGRP3, MTMR3, CLDN11, PPP3CA, SMARCA5, RNF144B, and AGO2 (Figure 1F and Supplementary 2).The prognostic role of these targets in MM was analyzed using previous cohorts (GSE9782 and GSE4581) [15], demonstrating that CLDN11, ICMT, MTMR3, RASGRP3, and SMARCA5 low expression are associated with poor prognosis of MM patients (Figure 2), whereas RAP1B, RCBTB1, PPP3CA, and AGO2 high expression are associated with poor prognosis of MM patients, and no statistic difference was observed between RNF144B expression and prognosis of MM patients (Figure S1).Moreover, TargetScan database suggested the presence of a binding site between miR-100-5p and the targets (Figure 2).These results indicate that miR-100-5p might be involved in the pathogenesis of MM by targeting CLDN11, ICMT, MTMR3, RASGRP3, and SMARCA5.

Functional enrichment analysis
To further investigate the cellular pathways associated with these five targets, we first predicated the main interactional proteins of them, the results were shown in Figure 3A.GO and KEGG enrichment were then performed to analyse the function and pathway enrichment of these main interactional proteins.All the enriched GO terms including molecular function (MF), biological process (BP), and cellular component (CC) categories, and KEGG pathways were displayed in Supplementary 3 and Supplementary 4, respectively.The top 10 GO terms of MF, BP, and CC were shown in Figure 3B, demonstrating that the interactional proteins were mainly enriched in protein binding term, plasma membrane term, and cytosol term.The top 20 KEGG enrichment pathways were shown in Figure 3C, and the top 10 KEGG enrichment pathways including enriched genes were shown in Table 3, containing inositol phosphate metabolism and phosphatidylinositol signaling system pathway.
MTMR3 inhibition impedes the effect of miR-100-5p suppression in living number, apoptosis, and metastasis of MM cells In addition, we inhibited the expression of miR-100-5p in both RPMI 8226 and U266 MM cells (Figure 4A).CCK-8 assay showed that miR-100-5p suppression declined the living number of MM cells (Figure 4B), and flow cytometry assay indicated that miR-100-5p inhibition enhanced apoptosis (Figure 4C).Western blot assay showed that the downregulation of miR-100-5p promoted the expression of Bax and E-cadherin proteins, and the cleave of caspase 3, while reducing the expression of Bcl-2, N-cadherin, and Vimentin proteins (Figure 4D).Furthermore, qPCR and western blot were performed to evaluate the expression of predicated five targets based on miR-100-5p inhibition, showing the expressive promotion of all five targets, especially MTMR3 (Figure 5).Thus, we selected MTMR3 for the subsequent study.The results showed that MTMR3 inhibition weakened the regulatory effect of miR-100-5p inhibition on living number (Figure 6A), apoptosis and metastasis (Figure 6B,C) in MM cells.

Discussion
Numerous studies have reported the effect of miR-100-5p in malignant tumor.For instance, Jakob et al. have found that the high expression of miR-100-5p is implicated with poor prognosis of oral squamous cell carcinoma [16]; Chen et al. have shown the overexpression of miR-100-5p in renal cell carcinoma and identified it as a diagnostic biomarker [17]; Liu et al. have demonstrated an elevation of miR-100-5p in ovarian cancer, and it is associated with the progression of ovarian cancer by targeting ependymin-related protein 1 [18].However, the function of miR-100-5p in MM was barely elucidated.In this work, differential expression analysis using high-throughput sequencing showed that compared to Control group, 32 miRNAs were overexpressed in MM group, especially miR-100-5p.miR-100-5p upregulation in MM patients were then verified in 21 samples using qPCR.ROC curve analysis indicated miR-100-5p as a diagnostic biomarker of MM.Although as highly conserved single-stranded small endogenous non-coding RNAs, miRNAs are very important genetic regulators, as evidenced by the fact that a single miRNA can direct entire cellular signaling pathways by targeting a broad spectrum of genes [19].In this study, to explore the potential biological regulatory effect of miR-100-5p in MM, we predicated the targets of miR-100-5p using miRanda, miRWalk, and PITA databases, showing that 10 target genes can be found in all of these three databases (intersection), and among them, CLDN11, ICMT, MTMR3, RASGRP3, and SMARCA5 low expression are associated with poor prognosis of MM patients.As previously reported, CLDN11 is a member of Claudin family, silenced in malignant melanoma, gastric cancer, bladder cancer, and oral leukoplakias, thereby leading to tumors progression [20].ICMT is an enzyme involved in the progress of post-translational modification, and its genetic ablation inhibited tumor formation of breast cancer cell lines with Ras mutation in a xenograft model [21].However, inactivation of ICMT enhanced the number of KRAS-driven intraepithelial neoplasias and promoted pancreatic ductal adenocarcinoma progression [22].MTMR3 was found to be overexpressed in breast cancer patients, and MTMR3 inhibition suppressed proliferation and autophagy of breast cancer cells [23].In contrast, Yan et al. suggested that MTMR3 was downregulated in glioma tumorigenesis, thereby promoting glioma cell proliferation and migration [24].As an activator of Ras, RASGRP3 overexpression was observed in both papillary thyroid cancer and breast cancer, and it may be a potential therapeutic target for these two cancers [25].As for SMARCA5, several studies have demonstrated its high expression in gastric cancer, acute leukemia, and non-small cell lung cancer, and SMARCA5 suppression targeted by miR-100 inhibited the progression of non-small cell lung cancer and glioblastoma [26][27][28][29].Collectively, all five targets show regulatory effects on multiple tumors.The distinguished function of one target on different tumors may be contributed to tumor heterogeneity and different molecular regulatory mechanisms.
Furthermore, protein network interaction analysis was performed to find out the major interacting proteins of these five targets and KEGG pathway analysis was performed to predicate the main cellular pathways these genes enriched.The results showed that the corresponding genes were obviously enriched in multiple pathways, especially inositol phosphate metabolism and phosphatidylinositol signaling system.Inositol phosphates are signaling molecules observed in all eukaryotes [30], and its metabolism involves several biological processes including but not limited to necroptosis [31], intracellular calcium signaling [32], and transcription [33].Wei et al. have found that inositol phosphate metabolism is one of the most impaction metabolic pathways in early breast cancer [34], and Zhu et al. have suggested inositol phosphate metabolism as one of the most influential pathways contributing to colorectal cancer [35].However, the influence of inositol phosphate metabolism in MM

Figure 1 .
Figure 1.MiRNA-seq data analysis and miR-100-5p expression verification.(A) A volcano plot was applied to visualize expression profiling between MM and control groups.The vertical black dotted lines correspond to a 2.0-fold (log2 scaled) increase and decrease, respectively.The horizontal black dotted refers to a p-value of 0.05 (-log10 scaled).Each red point represents a differentially expressed miRNA with statistical significance.(B) Number of upregulated and downregulated differentially expressed miRNAs in MM patients compared to control individual.(C) Hierarchical clustering indicates differences in miRNA expression profiling between the MM and control groups.(D) Validation experiment of miR-100-5p expression in MM patients.(E) Receiver operating characteristic curves of miR-100-5p.(F) MiR-100-5p targets predicated by miRanda, miRWalk, and PITA databases.

Figure 4 .
Figure 4. Function of miR-100-5p in MM cells.(A) qPCR was performed to detect the expression of miR-100-5p in MM cells after 24 h of transfection.(B) CCK-8 was performed to detect the living number of MM cells after 24, 48, and 72 h of transfection.(C) Flow cytometry was performed to detect apoptosis of MM cells after 24 h of transfection.(D) Western blot was performed to detect the expression of apoptosis-and metastasis-associated proteins in MM cells after 24 h of transfection.All Experiments were performed three times.***p < 0.001.

Figure 5 .
Figure 5.The targets of miR-100-5p expression in MM cells after transfection.(A) qPCR and western blot were performed to detect the expression of CLDN11, ICMT, MTMR3, RASGRP3, and SMARCA5 in MM cells after 24 h of transfection.All Experiments were performed three times.***p < 0.001 vs. miR-NC group.

Figure 6 .
Figure 6.Function of MTMR3 in miR-100-5p inhibited MM cells.(A) CCK-8 was performed to detect the living number of MM cells after 24, 48, and 72 h of transfection.(B) Flow cytometry was performed to detect apoptosis of MM cells after 24 h of transfection.(C) Western blot was performed to detect the expression of apoptosis-and metastasis-associated proteins in MM cells after 24 h of transfection.All Experiments were performed three times.***p < 0.001.

Table 1 .
Baseline characteristics of MM patients.

Table 2 .
Sample details from RNA-seq.