RBMX/hnRNP G has been recently acknowledged for its function in alternative splicing processes for many mRNA23. 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 mRNA10. 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 noise19. 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 genes15, 26. Previously, HOTAIR has been identified to contribute to poor prognosis, increased proliferation and metastasis in many cancers such as breast cancer15, pancreatic cancer16, colorectal cancer17 and HCC18. 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) motif13, 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 cancer19, gastric cancer22 and colorectal cancer20.
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 functions27. 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 survival19. 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 cancer20. 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 HCC6, 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 sorafenib28, 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 autophagy34, 35, 36. Other genes such as PSMD1037, ADBR238 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.
In recent years, different cancers have been observed to develop multidrug resistance, specifically ncRNA have been associated to contribute to such resistance. Specifically, the study by Zhou et al., which had proven that HOTAIR, a lncRNA, plays a vital role in cisplatin resistance to hepatocellular carcinoma15. They observed that HOTAIR induced MDR through STAT3 (signal transducer activated transcription), which in turn phosphorylated ABCB1. One type of MDR is actually due to ABCB1 (ATP-binding cassette sub-family B member) which pushes out the anticancer drugs out of the cells thereby reducing the intracellular drug39. Recently, many studies have also shed light on the role of another non-coding RNA microRNA (miR). Studies have indicated miRs such as miR-129-5p40, miR-36122 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.