In the present study, we have shown the expression pattern of OPN splice variants in GC. OPN-b was demonstrated as the dominant isoform in GC cell lines, and OPN-c was also elevated in most GC cell lines while barely detected in normal gastric cell line. In GC tissues, increased expression of OPN-b or OPN-c, but not of OPN-a, was associated with diverse clinicopathological features. In according with our observation, it has been reported that high OPN-b or OPN-c expression is correlated with adverse clinicopathological outcomes in both breast cancer and soft tissue sarcoma 27, 28
. Together with these findings in GC cells and tissues, we therefore postulate that the abnormal alternative splicing which results in elevated OPN-b or OPN-c expression may intensively occur during the progression of GC and then afford it more aggressive potentials.
We next clarified the diverse biological functions of three OPN splice variants through gain-of-function model in which GC cells stably overexpressed individual OPN isoform. We demonstrated that OPN variants presented the same subcellular distribution pattern in predominantly either cytoplasmic or nuclear compartment, which is consistent with the previous report16
. Besides, we found that three OPN splice variants all increased GC cell proliferation with no significant difference among them, which apparently indicates their similar proliferative competence in GC. After the functional heterogeneity of three OPN isoforms was further observed in both GC anti-apoptosis and metastasis, we also explored their different regulative effect on related downstream targets by overexpressing individual OPN isoform in MKN45 cell line since it presents the lowest endogenous level of each variant among our seven gastric cell lines.
It has been reported that OPN plays important role in preventing programmed cell death in response to pro-apoptotic signals such as oxidative stress stimuli 29
. In agreement with this, our study also demonstrated that both OPN-b and OPN-c could effectively protect GC against cells apoptosis which is induced by H2
. Moreover, OPN-b had stronger anti-apoptotic effect than OPN-c did, which is consistent with a previous report of three OPN isoforms in glioma 11
. However, no cell protection was observed in our study by OPN-a overexpression which has also been reported to prevent glioma cell from apoptosis 11
. Therefore, we indicate that the anti-apoptotic abilities of OPN splice variants are diverse in GC. Bcl-2 family members are known as crucial players involved in cell apoptotic process as they are able to regulate the assembly of apoptosome and result in subsequent caspase activation 30
. Our study further showed that OPN-b and OPN-c could both alter the balance between these pro- and anti- apoptotic proteins, thus leading to decreased procaspase cleavage and increased cell survival. Notably, OPN-b had an overall stronger pro-survival effect on the modification of this balance than OPN-c did. However, no altered levels of these apoptosis-related proteins could be found by OPN-a overexpression. Our study thus suggests that OPN splice variants exert diverse anti-apoptotic abilities by differently regulating Bcl-2 family proteins. Besides, it has been reported that both CD44v6 and CD44v7 which serve as OPN receptors on cell surfaces can coordinate the activation of downstream anti-apoptotic molecules and protect cancer cell against apoptosis 5, 31, 32
. Accordingly, our results demonstrated that overexpression of either OPN-b or OPN-c could lead to both elevated levels of CD44v6 and CD44v7, thus promoting consequent GC cell survival. Moreover, the most significant upregulation of CD44v7 which resulted from OPN-b overexpression might further explain the strongest anti-apoptotic effect of this isoform since CD44v7 plays a pivotal role in cell survival as previously reported 32
OPN has been implicated as an important mediator of tumor metastasis 33
. In our study, overexpression of each OPN protein isoform also promoted GC cell migration and invasion, which are two crucial processes in metastasis. It is worth mentioning that OPN-c elicited the strongest pro-metastatic potential among the three variants, thus highlighting its role in GC progression. To further understand the molecular basis involved in the pro-metastasis properties of OPN isoforms, we next detected the secretion of MMP-2 and uPa, both of which are closely related to the metastatic potential of GC 24, 25
. Meanwhile, secretion of IL-8 was explored because it can also act as pro-metastatic chemokine 26
. In our study, ectopic expression of each OPN isoform increased secretion of both MMP-2 and IL-8, with the strongest IL-8 upregulation induced by OPN-c overexpression. However, elevated secretion of uPa could only result from ectopic expression of OPN-c. Overall, our in vitro
assays suggest that OPN splice variants all promote GC metastasis through increased MMP-2 and IL-8 secretion, and that OPN-c exerts its most potential pro-metastatic effect among three variants via both elevation of uPa and strongest upregulation of IL-8. For further in vivo
metastasis assays, we demonstrated that both OPN-b and OPN-c, but not OPN-a, promoted hepatic metastasis in nude mice, and that OPN-c also represented the strongest pro-metastatic isoform. Those in vivo
observations of three OPN splice variants may attribute to the combination of their diverse anti-apoptotic and in vitro
pro-metastatic abilities. To our knowledge, this is the first study which has discussed the in vivo
metastatic potentials of three OPN isoforms.
Further loss-of-function experiment of three OPN variants would be a valuable complement to the study. However, it's technically difficult for us to downregulate each OPN splice variant both specifically and efficiently by RNA interference method, as the target sequences of each isoform which distinguish the one exclusively from the others were only limited at the specific junction of exons according to the difference among their mRNA structures 8
. Hence, to successfully design loss-of-function study and to possibly attain further therapeutic objective, OPN isoform-specific blocking antibodies need to be established in the future investigation.
As portrayed earlier in this study, there is an aberrant alternative splicing of OPN in the development of gastric neoplasm, which contributes to an aggressive profile of this cancer. Alternative splicing, i.e. the process of pre-mRNA splicing including the excision of introns and ligation of exons, can be activated by the proteins containing serine-arginine-rich sequences (SR proteins) 34
. In gastric tissues, it has been reported that the SR proteins exhibit upregulation and stimulation in response to Helicobacter pylori infection which induces approximately 75% of gastric cancer cases 35
. Although little is known about the exact mechanisms underlying the alteration in the splice site of OPN pre-mRNA, we suppose that H. pylori infection is involved as original stimulus. Further studies are necessary for the possible correlation between this bacterium contagion and aberrant expression profile of OPN splice variants in GC development.
Furthermore, it is worth exploring the possible mechanism for three OPN splice variants about their distinct functional activity in GC context. As compared with full length OPN (OPN-a), loss of exon 4 (OPN-c) or exon 5 (OPN-b) may result in altered protein structure and thus damage OPN physiological functions in normal gastric tissue. Concretely, two glutamine residues essential for transglutaminase cross-linking are present in sequences corresponding to exon 4. Therefore, OPN-a and OPN-b, but not OPN-c, can form polymeric OPN complexes which increase the stability of protein 36
. However, excision of exon 4 may make OPN-c more soluble and available for integrin receptors, thus resulting in its strong pro-metastatic activity through downstream enhanced signaling 37
. Accordingly, there is one cluster of phosphorylated serine/
threonine residues in sequences corresponding to exon 5 which is absent in OPN-b. As previously studied, tumors appear to express hypophosphorylated OPN which may contribute to aggressive cell behaviors 9
. Moreover, total protein phosphorylation status may be not as important as phosphorylation at specific site. Overall, we hypothesize that the phosphorylated residues in exon 5 may be essential to maintain OPN physiological activities, and that the deletion of exon 5 in OPN-b may therefore lead to its strong anti-apoptotic activity. However, further investigation will help elucidate the exact mechanism of altered OPN isoforms' profile and their functional diversity in GC context.
In conclusion, OPN-b is the dominant isoform in GC cell lines and the elevated expression of either OPN-b or OPN-c in GC tissues has their clinicopathological significance. Moreover, OPN-b exerts the strongest anti-apoptotic effect and OPN-c most potentially promotes GC metastasis. Overall, our study suggests that OPN splice variants have different clinical and biological features in GC. Thus, exploring the roles of OPN splice variants in GC will be a novel direction to develop both diagnostic and therapeutic approaches.