RBMX promotes hepatocellular carcinoma progression and sorafenib resistance by stabilizing BLACAT1 CURRENT STATUS: REVIEW

Background Hepatocellular carcinoma is one of the top five causes of cancer death. The interaction of RNA binding proteins and long no coding RNA play vital role in malignant tumor progression, and even contribute to chemoresistance. Method RBMX and Lnc BLACAT1 were measured in HCC patient’s tissue and commercial cell lines (Huh7 and Hep3B). And there relationship was verified by Co-IP assay and Overexpression experiment. Furthermore, the cell proliferation, colony formation and drug resistant assay were performed for effect evaluation of RBMX and BLACAT1in HCC cell lines. Result Both overexpression and positive correlation between RBMX and BLACAT1 were validated in HCC cell lines. Co-IP analysis also shows BLACAT1 could be specially bond by RBMX and increased the expression of BLACAT1. This mechanism of action is beneficial for cancer cells proliferation, anti-apoptotic, and colony formation with sorafenib treatment. Further, the autophagy level and cancer cell stemness were also improved when RBMX/BLACAT1 upregulated. Conclusion Our study indicated that hepatoma cells can improve their proliferation, colony ability and autophagy by RBMX stabilizing BLACAT1 expression then promote HCC development and drug resistance, hence, RBMX could be considered as novel therapeutic target for HCC treatment strategies.

advanced stages of HCC are very limited due to the many underlying liver diseases which are associated with carcinoma 2 . Sorafenib (Nexaver, Bayer HealthCare Pharmaceuticals) is a drug that mechanistically inhibits the serine threonine kinase Raf and tyrosine kinase activity of vascular endothelial factor receptors, has been known to inhibit tumor cell proliferation and angiogenesis in different tumor models, including HCC. Sorafenib treatment has been considered as first option for patients with advanced stages of HCC, with the severity of the disease being high most complications arise from the carcinoma associated liver diseases 3, 4, 5 . Sorafenib in clinical studies such as the SHARP trial 6 and Asia-pacific trial 7 have been able to improve the survival rate of patients by 2.5 to 3 months in patients compared to the placebo group. HCC being a hugely heterogenetic disease, many patients seemed to show primary resistance to the sorafenib treatment 8 . The exact mechanism behind such resistance is still unclear.
In the current study, we observed high expression of RBMX gene (RNA binding motif) in HCC cell lines but additionally more increased expression in HCC-sorafenib resistant (SR) lines. Hence, we explore the potential role of RBMX in HCC progression and sorafenib resistance. Human proteome expression is incomplete without appropriate splicing of the nuclear mRNA and such splicing is carried out by many ribonucleoproteins (hnRNPs) which bind with the nascent mRNA and form complexes 9 . Among these, hnRNP G is coded by the RBMX gene and is relatively the least abundant but implicated in splicing of many vital mRNA 10 . Further, they play a important role in either addition or exemption of exons in the mRNA for many proteins. Recent studies have shown that hnRNP G associates itself with most units of RNA polymerase II hence being a basic component of general transcription process 11 .
Another study had shown that RBMX binds with satellite I mRNA during mitotic phase, and lack of RBMX led to defective centromere cohesion and abnormal segregation, indicating its very crucial role during M phase 12 .
A study by Heinrich et al., 13

mRNA Stability Assay
Huh7 or Hep3B cells transfected with scr or shRBMX-1(shRBMX) expression vector and the stability of BLACAT1 mRNA variants were examined by treating the cells with actinomycin D (Sigma). At different time points, (0, 15, 30, 60, and 120 min) RNA was isolated qRT-PCR were performed, as described above. Half-life (t1⁄2) of BLACAT1 mRNA variants were calculated as the time required for each mRNA variant to reduce to 50% after actinomycin D treatment from its initial abundance at time 0 min. Halflives ( t1⁄2 ) were determined by nonlinear regression analysis.

2.15Database and software predictions
In order to get a wider range of evidence, the dysregulated gene expression of RBMX was further verified by The Cancer Genome Atlas (TCGA) database (n = 371); also the survival correlation (http://ualcan.path.uab.edu/analysis.html). CatRAPID program were used for interaction prediction (http://service.tartaglialab.com/page/catrapid_ group).

Statistical Analysis
The data are presented as the means ± standard deviations (SD). Tukey's test or Student's t -test for the unpaired results was used to evaluate the differences among more than three groups or between two groups, respectively. Differences were considered significant for values of p < 0.05.

RBMX is overexpressed in HCC tissues and HCC cell lines
To confirm the expression of RBMX in tumors, transcript levels were compared between the tumor and normal tissues. Based on the evidence from TCGA databases, RBMX transcript levels were observed to be highly upregulated when compared to the normal tissues ( Fig. 1A), as well as the association of clinicopathological characteristics of HCC patients with RBMX expression was showed in Table 1. And that, it was also observed the survivability of patients with high expression of RBMX had poor prognosis when compared to the patients with low or medium expression of RBMX (Fig. 1B). In six individual patient tumor samples western blotting analysis was performed with comparisons to the normal tissue samples (Fig. 1C). It was clear that there was significantly higher expression of RBMX in the patient samples, which was also observed in the mRNA level (Fig. 1D). Immunohistochemistry analysis was performed in hepatocellular carcinoma (HCC) patient tissues and consistent results were evident that RBMX was highly expressed (Fig. 1E). Commercially available hepatic cell lines such as LO2, HepG2, Huh-7, SMMC7721, Hep3B, HCCLM3 and MHCC97H were checked for RBMX expression ( Fig. 1F, G). Among these, Huh-7 displayed the highest expression of RBMX followed by HepG2. And, Huh-7 or Hep3B cells were choose for further experiments.

Crucial role of RBMX in HCC cell's proliferation and apoptosis
To understand the effect of RBMX in HCC progression, silence or overexpression experiments were performed in two different cell lines first. RBMX gene was silenced in Huh-7 cells, and silencing efficiency was confirmed using qRT-PCR analysis and western blotting analysis, and it was evident that the shRBMX-1 and shRBMX-2 had a much lower RBMX expression compared to the non-functional control shRNA (scr) ( Fig. 2A). RBMX overexpression assay was performed in Hep3B cells, and overexpressing efficiency was also verified in (Fig. 2B). Then, proliferation assays were performed with these cells using cell counting kit. The results indicated that RBMX silenced cells have decreased proliferation (Fig. 2C), on the contrary, RBMX overexpressed cells displayed a higher proliferation ability (Fig. 2D). Further, colony formation capacity shows shRBMX Huh-7 cells had lesser colony forming units when compared to scr (Fig. 2E), adversely, Hep3B cell's colony units was significant increased when overexpressing RBMX (Fig. 2F), this results showing a strong positive correlation between RBMX and proliferation. TUNEL assay were performed to evaluate the cells apoptosis in RBMX overexpressed and silenced cells. It was evident that the cells which RBMX were silenced had very high number of apoptotic cells when compared to the control (Fig. 2G), contrarily, the dead cells is minor in RBMX overexpressed Hep3B cells (Fig. 2H). Next, the data was further verified using flow cytometer analysis for RBMX silencing (Fig. 2I). Taken together, these date demonstrated that RBMX could facilitate HCC cells proliferation and inhibit cell apoptotic.

RBMX promote tumor growth in model
Next, the role of RBMX in tumor growth was studied. For this,shRBMX Huh-7 cells were injected into the dorsal flanks of nu/nu mice. The tumor volumes were subsequently measured every week using digital calipers. It was evident that the tumor size increased significantly from day 7 to day 35 in the scr group (Fig. 3A, B). But the development of tumors was significantly much slower in shRBMX group, when compared to the control group at the same time points. At day 35 the mice were sacrificed, and the tumor weight was measured. It was clear that the shRBMX group tumors were significantly smaller when compared to the scr group (Fig. 3A, C). Further, these samples were subjected to immunohistochemistry staining. Silencing efficiency was confirmed with RBMX immunohistochemistry staining, where in the shRBMX group tumor section displayed less to no positive cells for RBMX antibody (Fig. 3D right). Additionally, hematoxylin and eosin staining of the tumor sections from the control group showed distinct cells with clear darker nuclei typical of cancerous cells, but shRBMX group showed lesser nuclei (Fig. 3D left), indicating a decrease in tumorous cells. Ki67 staining was also performed which indicated more proliferative cells present by the darker nuclei in the scr group when compared to the shRBMX group. Finally, TUNEL assay of these sections displayed higher apoptotic cells in the shRBMX group compared to the scr (Fig. 3D middle). Taken together, these dates indicated that RBMX promote tumor growth by improve cells proliferation and decrease apoptotic.

RBMX contributes to HCC cell lines sorafenib resistance
To further understand the role of RBMX in chemoresistance, sorafenib resistance HCC lines were established as previous (Huh7-SR, Hep3B-SR), and CCK-8 assay was performed for resistant evaluation. As show in Fig. 4A B, There have a huge improvement of sorafenib resistance ability.
Then, the expression of RBMX was measured in sorafenib resistance cell lines. We can observe that, the level of RBMX mRNA and protein significantly increased in Huh7-SR/Hep3B-SR compare to Huh7/Hep3B native cell. More than that, the expression level of RBMX was further enhanced under sorafenib treatment (Fig. 4C, D). Further, cell viability assay was performed for evaluation of RBMX function in RBMX overexpressing or silenced cells. With increasing concentrations (0-20 µM) of sorafenib, it was evident that the cell relative viability of Huh7/Huh7-SR cells which RBMX have been silenced significantly decreased (Fig. 4E). But RBMX overexpressing cells, Hep3B/Hep3B-SR, had higher cell viability than empty vector (Fig. 4G), thus indicating a important role of RBMX in resistance to sorafenib. Apoptosis level was also checked using flow cytometry. It was observed that the percentage of apoptotic cells in different RBMX expression level is consistent with cell viability (Fig. 4F, H). Simultaneously, to identify specific indicators of drug resistance and its association with RBMX, we checked the presence of ABCB1 (ATP Binding Cassette Subfamily B Member 1, a vital indicator of multidrug resistance) in HCCs (Fig. 4I). Western blotting data indicated that increased expression of RBMX did increase ABCB1 expression. And, ABCB1 expression decreased significantly in both Huh7/Huh7-SR and Hep3B/Hep3B-SR cell lines when RBMX was silenced. Simultaneously, to further understand the correlation of RBMX and apoptosis, caspase 3 expression was checked. From the data, it was clear that caspase-3 expression was reduced while RBMX was upregulated (Fig. 4I), but when RBMX was silenced caspase-3 expression was highly upregulated, confirming our previous study of RBMX's role in cell proliferation. Based on these data we could declare that RBMX plays a key role in sorafenib resistance of HCCs.

Autophagy is a consequence of RBMX mediates sorafenib resistance
Further experiments were also performed to understand the mechanism of how RBMX participate in sorafenib resistance. Interestingly, it was observed that the cells autophagic maker LC3 significantly increased in sorafenib resistant cells and RBMX up-regulated cells, there have an opposite expression levels in HCC normal cells and RBMX down-regulated cells (Fig. 5A-D). Western blotting analysis indicated that the LC3 II was significantly increased and p62 was downregulated in the presence of RBMX, but when RBMX was silenced then LC3 II and p62 were significantly opposited (Fig. 5E, F).
Thus, the above-mentioned experiments specifically indicated the roles of RBMX mediate sorafenib resistance of hepatocellular carcinoma cells by activating autophagy.

RNA-binding protein RBMX binds and stabilizes BLACAT1
Many studies have showed the chemotherapeutics resistant ability of BLACAT1in multi-cancers, but the specific mechanism of action is unclear. For this concern, we talk about the relationship between RBMX and BLACAT1 in HCCs. First of all, BLACAT1 level was measured in HCC patient tissues and commercial HCC lines (Fig. 6A, B), and results show that there have a positive correlation between them (Fig. 6C). As mentioned previously, another study had identified the hnRNP G's RNA-recognition motif binds to a loose consensus sequence containing a CC(A/C) motif (13). In this study, we identified many similar CCAG consensus sequence by comprehensive mutational analysis of the RBMX-binding sites in the lncBLACAT1.
We identified a total of 29 such sites in BLACAT1 sequence (Fig. 6D). More than that, CatRAPID online algorithm also performed for RNA-protein interaction prediction (Fig. 6E). Of interest, this program revealed a higher interaction DP value, reach up to 98%.Next, RNA immunoprecipitation assays were go ahead for interaction validation. RIP analysis of the samples showed the RBMX presences in both the Huh7 and Hep3B cells, which contain BLACAT1, thus indicating a strong interaction between BLACAT1 and RBMX (Fig. 6F, G). Additionally, RNA immunoprecipitation analysis followed by qRT-PCR also showed that sample precipitated with RBMX had a high fold expression of BLACAT1 (Fig. 6H, I).
Experiments to understand the stability of BLACAT1 mRNA in the absence of RBMX were studied. Cells were either transfected with scr or shRBMX and then treated with actinomycin D to block de novo transcription. Further, the levels of BLACAT1 were quantified using qRT-PCR and normalization was performed to 18S rRNA levels. It's obvious that, BLACAT1 mRNA had decayed to almost 60% within performed for further verification (Fig. 6J, K). The expression level of BLACAT1 was distinctly increased with the overexpression of RBMX in all these cells, and when RBMX was silenced, BLACAT1 was downregulated. Taken together, these date indicating a strong interaction between RBMX and BLACAT1.

RBMX knockdown inhibited BLACAT1-induced cell proliferation
Overexpression studies were further used to evaluate the effect of BLACAT1 on cell proliferation. The date shows, it's evident that BLACAT1 overexpression (Fig. 7A) in Huh7 cells increased the proliferation of HCC cells (Fig. 7B), also with an increase in colony forming capacity (Fig. 7C). The same trend of cells proliferation (Fig. 7D) and colony formation ability (Fig. 7E) also appeared in Hep3B cell line which transfected with BLACAT1 overexpressed vector (Fig. 7F). However, Huh7 cells and Hep3B cells which BLACAT1 gene overexpressed (Fig. 7G, J) lost their both proliferation (Fig. 7H, K) and colony forming capacity (Fig. 7I, L) when RBMX was silenced. In previous experiments, we had shown that RBMX increases cell viability; here we show that BLACAT1 can also increase cell viability but only in the presence of RBMX. This indicates BLACAT1's proliferation capacity is dependent on RBMX.

RBMX knockdown inhibited BLACAT1-induced autophagy and sorafenib resistance of HCC cancer cells in vitro.
To further understand the role of BLACAT1 in RBMX mediated sorafenib resistance, Huh7 and Huh7-SR cells with overexpression of BLACAT1 and treatment with sorafenib were performed. Initially, we found that the Huh7 cell which BLACAT1 overexpressed remained higher cell viability in concentration gradually increased sorafenib treatment, than the empty vector, indicating that BLACAT1 itself could contribute for the resistance to sorafenib (Fig. 8A). But, the cells viability decreased significantly with sorafenib treatment when RBMX was efficiently silenced, even at as low as 2.5 µM concentrations, making the cells highly susceptible to sorafenib (Fig. 8B). Further, the multidrug resistant marker (ABCB1) and apoptotic marker (caspase 3) were checked in the BLACAT1 over expressed and RBMX silenced cells. The date showed that ABCB1 expression raised and caspase3 declined clearly respectively, but the silence of RBMX reversed the expression levels of both (Fig. 8C), which indicated BLACAT1 similar to RBMX seemed to contribute to drug resistance in HCC. Similarly, the same experiments were performed in Huh7-SR cells which have uniform treatment as Huh7 cells. As we expected, there showed consistent consequence in cells viability, ABCB1 and caspase3 expression in RBMX silenced or not groups when BLACAT1 was overexpressed (Fig. 8D-F). Thus, overexpression of BLACAT1 increases cell's (both Huh7and Huh7-SR cell lines) viability but the consequence were reversed as RBMX knock down. This indicated that RBMX binding to BLACAT1 is vital for the sorafenib resistance. Additionally, cells autophagy was detected. We observed that BLACAT1 overexpression seems to promote LC3 expression (Fig. 8G, H), but LC3 expression seems to be declined when RBMX is silenced even in the presence of BLACAT1. Specifically, although LC3 II expression seems to be upregulated in the BLACAT1 overexpression system, however it is similar to vector control when RBMX is silenced,but LC3 I expression remains the same throughout (Fig. 8I). Also P62, another autophagy marker, seems to be downregulated when BLACAT1 is upregulated, but when RBMX is silenced they seem to be upregulated (Fig. 8H). Hence, silencing of RBMX significantly affect the drug resistance ability by upregulating apoptosis and downregulating autophagy. Taken together, these date indicating the vital role of RBMX in regulating BLACAT1 and sorafenib resistance in HCC cells.

Discussion
RBMX/hnRNP G has been recently acknowledged for its function in alternative splicing processes for many mRNA 23 . Interestingly, these functions vary between activation or inhibition depending on the mRNA. A study by Nasim et al., 10 had shown that RBMX interacts with a splicing activator protein called hTra2β, and they together antagonistically are involved in inclusion or exclusion of exons in many mRNA. This particular procedure allows certain genes to be either suppressed or expressed in a particular cell type. Another study identified estrogen receptor alpha (ERa) 24 expression is regulated by hnRNP G and a splicing regulator/opponent HTRA2-BETA1. In this study, hnRNPG was required for the inhibition of the splicing of ERa exon 7 by HTRA2-BETA1. Surprisingly, this inclusion and higher expression of hnRNP G aided in improved prognosis and survival in endometrial cancer. In contrary, in our study we observed increased expression of RBMX led to poor prognosis in HCC patients (Fig. 1B).
Additionally, we observed overexpression of RBMX led to an increased proliferation and colony formation capacity in HCC cell lines (Fig. 2). This alternative role could be well explained, because hnRNP G has varying roles depending on the target mRNA 10 . There are many such studies showing hnRNP G to have multiple splicing roles in mRNA of proteins such as survival motor neuron SMN2, Tau and dystrophin proteins 10, 25 .
In our study, we identified one such mRNA which seems to be regulated by RBMX, which is BLACAT1 (Fig. 6). BLACAT1 which is also known linc-UBC1 is a long intergenic non-coding RNA, they are among the recently identified class of ncRNA and have been neglected for years as just non-essential background noise 19 . In recent years many of such lncRNA are being brought into limelight for their role in cancer. One such lncRNA is HOX transcript antisense RNA (HOTAIR) which seems to be interacting with polycomb repressive complex and in turn upregulate the trimethylation of histones, which subsequently lead to downregulation of many genes 15, 26 . Previously, HOTAIR has been identified to contribute to poor prognosis, increased proliferation and metastasis in many cancers such as breast cancer 15 , pancreatic cancer 16 , colorectal cancer 17 and HCC 18 . In our study, we were very interested to identify the relationship between RBMX and BLACAT1. Previously, a study identified the RNA recognition motif of RBMX binds to a consensus sequence of CC(A/C) motif 13 , and in this study we identified BLACAT1 has total 29 such sites in BLACAT1 sequence (Fig. 6D). We also identified that BLACAT1 binds and interacts with RBMX through immunoprecipitation studies (Fig. 6F, G). It was also clear that RBMX not only binds to BLACAT1 (Fig. 6H, I) but positively upregulates the expression of BLACAT1 (Fig. 6J, K). Previous studies have shown BLACAT1 to be upregulated in bladder cancer 19 , gastric cancer 22 and colorectal cancer 20 .
From our evidence, RBMX also seems to bind and stabilize BLACAT1, as we observed post silencing of RBMX, BLACAT1 decayed quickly (Fig. 6J, K). This indicates the strong interactive role between RBMX and BLACAT1. It was also evident that lack of RBMX and in turn BLACAT1 seems to upregulate apoptosis, decrease proliferation, and also decrease autophagy in HCC cell lines (Fig. 8). There have been other studies where in binding of certain proteins are necessary to stabilize them, one study had shown the RNA binding protein known as "quaking" was necessary to stabilize SIRT2 mRNA for it to perform its functions 27 . Understanding the potential functions of BLACAT1 leads to one of the earliest studies which identified its role in bladder cancer due to its interaction with polycomb repressive complex 1 and has been associated with lymph node metastasis and poor overall survival 19 .
BLACAT1 has previously also been associated to increased cell proliferation due to its ability to bind with EZH2 and suppresses p15, thereby disrupting the cell cycle in colorectal cancer 20 . Hence, BLACAT1 could potentially be the chief contributing factor for tumorigenesis but it was evident that RBMX was regulating the BLACAT1 in HCC. When cells were overexpressed for BLACAT1 and treated with sorafenib, the proliferation of these cells were higher than the empty vector. But when these same cells were silenced for RBMX and treated with sorafenib, the cell viability decreased significantly in HCC and HCC-SR cells (Fig. 8).
Another interesting observation from our data was that RBMX also contributed to multidrug resistance in HCC. In this study, we considered one of the important treatment strategy sorafenib that has been used to treat advanced stages of HCC 6, 7 . In our study, we observed sorafenib resistant (SR) cell lines also had very high expression of RBMX (Fig. 4C). Treatment of sorafenib had very less effect on decreasing the cell viability (Fig. 4E, G) of HCC-SR cell lines. But silencing of RBMX (Fig. 4E) aided in improving the sensitivity of sorafenib significantly, thereby indicating a strong role of RBMX in sorafenib resistance. Additionally, we observed in both the HCC and HCC-SR lines, sorafenib dependent apoptotic activity increased significantly after silencing of RBMX (Fig. 4E). There are various studies which have indicated that sorafenib can activate protective autophagic response in HCCs, and detailed experiments where in various pharmacological inhibitors such as bafilomycin 1, chloroquine or knockdown of various autophagic genes such as beclin or atg5 have been indicated to improve the cytotoxic effect of sorafenib 28, 29, 30, 31, 32, 33 . Another study had shown CD24 to be highly expressed in HCC, which in turn activates protein phosphatase 2, this additionally seems to activate mTOR/AKT pathway which contributes to autophagy 34, 35, 36 . Other genes such as PSMD10 37 , ADBR2 38 also have shown to promote HCC progression through their regulation of autophagy. In our studies too, we observed autophagy to be highly upregulated post treatment with sorafenib in the resistant cell lines (Fig. 5A). But when RBMX was silenced, autophagy was highly downregulated with decrease in proliferation, thus overcoming sorafenib associated protective mechanism in HCC-SR. causes chemo-resistance in gastric cancer through activation of ABCB1. In our study too, we observed ABCB1 to be upregulated in sorafenib resistant cells, specifically after treatment with sorafenib. But once these cells were silenced for RBMX, ABCB1 expression decreased significantly (Fig. 8D).
Based on our studies and multiple evidences from literatures, we could conclude that RBMX through BLACAT1 induces tumorigenesis, which was observed by increasing proliferation and decreasing apoptosis. Further it also regulates MDR through ABCB1 and autophagy (LC3). Our results could contribute in development of therapeutic strategies for advanced stages of HCC and other cancers whose treatment is a limited to specific type of therapeutic strategy.

Conclusion
Overall, the results in this study indicated that under the mechanism of RBMX stabilize and promote BLACAT1 expression, hepatoma cells can increase their proliferation and colony ability then promote HCC development. Furthermore, the autophagy is also improved when co-incubate with sorafenib, contribute to chemotherapy resistance. Hence, RBMX could be considered as novel therapeutic target for HCC treatment strategies.

Ethics approval and consent to participate
All patients signed the informed consent and the study was approved by Institute Research Ethics Committee of the Seventh People's Hospital of Shanghai University of TCM.

Consent for publication
Not applicable

Availability of data and materials
Not applicable

Competing interests
The authors declare that they have no conflict of interest.

Funding
The study was supported by grants from National Natural Science Foundation

Authors' contributions
All the authors contribute to design and discussion of this study. Furthermore, Y S and S H contribute to article writing; X M and M Z contribute to experiments performance; J Z and G W contribute to date analysis and figure construction; Y Y and W X provide guidance and assistance throughout the process.