Long Non-Coding RNA FAM157C Contributed to Clonal Proliferation in Paroxysmal Nocturnal Hemoglobinuria

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal disease of hematopoietic stem cells. However, the mechanism of proliferative advantage of PNH clone is unclear. Long noncoding RNAs (LncRNAs) have a wide range of biological functions, including regulation of gene expression, cell differentiation, and proliferation, while its role in PNH remains unclear. Methods: In our study, CD59 (cid:0) and CD59 + granulocytes and monocytes from 5 PNH patients were sorted, and LncRNAs and mRNAs were detected by RNA sequencing. The proliferation-related NF-κB pathway was focused on. A total of 8 mRNAs and 5 LncRNAs were veried by qRT-PCR, and analyzed the correlation with clinical data. Meanwhile, the function of LncRNA was studied. Results: LncRNA FAM157C were veried to be upregulated in PNH clone cells, which were positively correlated with LDH level and CD59 - granulated and monocytes cells ratio. After knockdown of FAM157C gene in PIGA-KO-THP-1 cell line, we found that the cells were blocked in G0/G1 phase and S phase, and the apoptosis rate increased, while the proliferation ability decreased. Conclusions: LncRNA FAM157C was proved to promote PNH clone proliferation, which is the rst time to explore the role of LncRNAs in PNH. in the anti-apoptotic process 27 The studies also found that PIG-A mutations disrupt lipid raft formation of cell membrane, this change passivation for promoting apoptosis signals or growth inhibition 28 . The theory of secondary genetic mutations has been reported since the 1970s 21 . Additional mutant genes such as HMGA2 , WT1 31 , TET2 32 and RBPJ reported in PNH patients. Most of PNH can carry additional mutations and these secondary strikes.


Introduction
Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal disorder of haematopoietic stem cells PIGA mutation leads to impaired glycosylphosphatidylinositol (GPI) synthesis, resulting in the loss of GPI anchored protein (GPI-AP) on the cell surface (such as CD59, CD55), resulting in the destruction of the increased sensitivity of the blood cells to the activation of complement, and the main clinical manifestations are chronic intravascular hemolysis, bone marrow failure, high risk of thrombosis, etc 1 2 3 . The PIG-A mutation alone is necessary but insu cient to explain PNH clonal expansion. PNH clones do not self-renew and survive for only 3-4 months 4 . In order to cause PNH, PIGA mutations must achieve proliferative advantages. However, the mechanism of proliferative advantage of PNH clone is not clear until now.
Long noncoding RNAs (LncRNAs) range from 200 nt up to ~ 100 kb in length which do not encode proteins and are located in the nucleus or cytoplasm 5 6 . It is now well recognized that more than 75% of the human genome is functional and encodes large numbers of LncRNAs 7 . LncRNAs have a wide range of biological functions, including regulation of gene expression, such as chromosome dosage compensation, imprinting, epigenetic regulation, nuclear transport, transcription, mRNA splicing and translation; as well as cell differentiation, cell proliferation and substance metabolism 8 9 . In the past decade, the abnormal expression of LncRNAs have been proved to be involved in the pathogenesis of many diseases, including tumors, metabolic diseases and cardiovascular diseases 10 11 12 . At present, studies on the mechanism of PNH clone proliferation are mainly focused on protein-coding genes, the function and clinical signi cance of LncRNAs in the PNH remain unknown. The purpose of this study was to investigate the role of LncRNAs in PNH clone proliferation. 2.2 Cell sorting by ow cytometry Ten milliliter peripheral blood were taken in ethylenediaminetetraacetic acid (EDTA) anticoagulant tube from PNH patients. Firstly, the supernatant was discarded after centrifugation (3 g, 5 minutes), and the remaining blood was separated into ve centrifugal tubes. After 15 minutes, the erythrocyte lysate was centrifuged again and washed with PBS. Anti-CD59 FITC (BD PharMingen, USA) was added for 20 min in dark room.

RNA sequencing and analysis
After obtain CD59and CD59 + granulocytes and monocytes, quality of total RNA was extracted from PNH patients. RNA-seq analysis was performed by Beijing Novogene Bioinformatics Technology Co., LTD (Appendices 1).

qRT-PCR
Total RNA was extracted from CD59cells by RNeasy kit (Takara Bio Inc, RR420A, Japan). cDNA was synthesized from 1 µg of total RNA using a reverse transcription kit (Takara, Japan) and puri ed with the  Table 2. The BIO-RAD CFX MANAGER software was used to analyze the melting curve and the ampli cation curve (Quantitative curve), and the Ct values of each group were read. The relative quantitative multiplier of each group (relative fold, RF) was expressed by 2 − ΔΔCt value and used for statistical analysis.   incubator followed by measurement of the absorbance at wavelength of 450 nm.

Cell apoptosis assay
Apoptosis was determined by translocation of phosphatidylserine to the cell surface using an Annexin V-PE and 7-ADD apoptosis detection kit (BD PMG). The stable knockdown LncRNA FAM157C PIGA-KO-THP-1 cells and its negative control cells were harvested and washed twice in cold PBS, and re-suspended in Annexin V-PE and 7-ADD for 30 min in the dark. Cell apoptosis was analyzed by using Cell Quest software on a Flow Cytometer (Beckman).

Cell cycle analysis
The cells were rst harvested after 72 hours of transfection and the cell suspension was then digested.
Afterwards, the cells were xed with ethanol (70%) for 4 h at 4 °C and the supernatant was then discarded, followed by incubation with an RNA enzyme containing iodide (PI, BD PMG). After the cells were washed with PBS three times, the cell cycle was detected by using Flow Cytometer (Beckman), and data analysis was conducted through Kaluza. The experiment was repeated three times.

Statistical analysis
GraphPad Prism5 statistical software was used for statistical analysis. Results were expressed as mean ± standard deviations. The independent sample mean comparison had been done using the t-test (for data with normal distribution) and nonparametric test (for data without normal distribution). Spearman's correlation analysis was used to evaluate the association between qualitative variables. A value of P < 0.05 was considered statistically signi cant.

Some abnormal LncRNAs and mRNAs were sorted by RNA-seq in PNH clone
For transcriptome pro ling of CD59cells and CD59 + cells in PNH patients, RNA-seq was performed in CD59 and CD59 + granulocytes and monocytes from 5 PNH patients screening. Transcription analysis revealed that 742 upregulated and 1376 downregulated LncRNAs, and 3276 upregulated and 213 downregulated mRNAs ( Fig. 2A, B).
The 173 upregulated mRNAs which FPKM > 5 and over 3 patients were screened. Then we screened 30 mRNAs related to proliferation, apoptosis and thrombosis. The 7 upregulated LncRNAs which FPKM > 5 and over 3 patients were screened (Appendices 2). The expression of upregulated mRNAs and LncRNAs in 5 PNH patients were showed by venn diagrams (Fig. 2C).
We also observed the cell cycle, and the results showed that the proportion of G0/G1 phase and S phase

Discussion
In recent years, studies have found that PIG-A mutations can also be detected in normal people, accounting for about 10%. However, no PNH clones and proliferation have been found, and no clinical symptoms of PNH 14 . In Shin TH et al 2019, the data gathered about PNH macaque model using CRISPR/Cas9 technology to create model of hematologic disease based on the near phylogenetic/functional similarity between macaque and human was shown that there was no intrinsic clonal ampli cation of PNH-HSPCs 15 . At present, studies reported that immune-escape characteristics 16 17 18 , anti-apoptotic properties 19 20 , and second gene mutations 21 22 23 may be involved in the ampli cation of PNH clones. T-lymphocyte immune attack GPI + HSC, and GPI − HSC is less vulnerable to attack. Studies have found that PNH cells are protected from NK/T effector cells due to the lack of GPI-anchored cytomegalovirus ul-16 binding protein (ULBPs) or CD1d restriction 24 25 . CD109 has been reported to be a protein in GPI-APs, a TGF-co-receptor, which plays a key role in inhibiting TGF-signal-mediated erythrocyte differentiation. The lack of CD109 may make PIGA-mutated HSPCs more sensitive to TGF-β, leading to more easy differentiation of mutant erythroid progenitors into mature erythrocytes 26 . The studies found that PNH clone cells have anti-apoptotic properties. In addition, Bcl-2, Bcl-XL, Bag-1, McL-1 and other anti-apoptotic genes were signi cantly increased in PNH patients, and played an important role in the anti-apoptotic process 27 . The studies also found that PIG-A mutations disrupt lipid raft formation of cell membrane, this change passivation for promoting apoptosis signals or growth inhibition 28 . The theory of secondary genetic mutations has been reported since the 1970s 21 . Additional mutant genes such as HMGA2 29 30 , WT1 31 , TET2 32 and RBPJ 33 have been reported in PNH patients. Most cases of PNH can carry additional mutations and these mutations are secondary strikes. PIGA mutation is the initial mutation, the nature of PNH is a single gene disease, and its clinical manifestations are mainly determined by PIGA mutations rather than myeloid gene mutations 34 . However, various theories cannot explain all the pathological mechanisms, and PNH clones must be involved in other mechanisms to gain proliferation advantage in patients.
Although LncRNAs do not encode proteins, they play an important role in cell proliferation and differentiation, and are widely studied in oncologic diseases. Not only solid tumors, but also non-solid tumors and even autoimmune diseases have been studied. The LncRNAs LOC101928834, H19, WT1-AS, TCL6, LEF1-AS1, EPB41L4A-AS1, PVT1, GAS5 and ZFAS were found relevant to (myelodysplastic syndromes (MDS) pathogenesis and outcome 35 36 . Many LncRNAs such as MALAT1, GAS5, DLEU2, H19 and so on were reported in diagnosis ang progression of multiple myeloma (MM) 37 38 . The LncRNAs HOTAIR, LincRNA-p21, LncRNA H19 and MALAT1 play important role in clinical diagnosis ang progression of Rheumatoid arthritis (RA) 39 . The mechanism of action of LncRNA is complex. LncRNAs can interact with DNA, RNA, or protein.
In our study, the results of RNA sequencing in PNH patients showed a large number of differentially expressed LncRNAs and mRNAs, many of which were involved in cell proliferation, thrombosis, etc. Such results gave us a lot of information, LncRNAs may play an important role in PNH clone proliferation. By veri cation, we found that the level of LncRNA FAM157C in CD59 − cells sigini cantly increased, and was positively correlated with the LDH levels and CD59 − granulated and monocytes cells ratio. After knockdown of FAM157C gene, it was found that the cells were blocked in G0/G1 phase and S phase, the apoptosis rate increased, the proliferation ability decreased. The FAM157C was expressed in bone marrow, spleen and other organs, and its function has not been reported in the literature. The results of our experiment suggested that FAM157C gene may promote PNH clone proliferation. The mechanism remains to be further studied.
Declarations Figure 1 : The cells of PNH patients were sorted by Flow cytometry to obtain CD59 and CD59+ granulocytes and monocytes. B: Sorting purity of the CD59 and CD59+ granulocytes and monocytes. The sorting purity is about 90%.