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Host miRNAs and RBPs
[45]
Multiple miRNAs have been implicated in the control of PD-L1 expression in GC . Some miRNAs,
such as miR-152 and miR-200, target the 3’-UTR of PD-L1 directly. Other miRNAs, such as miR-19a and
miR-19b, affect PD-L1 levels indirectly, by targeting signaling pathways or transcription factors that
regulate PD-L1 expression [45-48] .

EBVaGC has been reported to exhibit a distinct host miRNA expression profile from other GC
subtypes [4,49,50] . Differentially expressed miRNAs include ones that have been independently shown to
[48]
target PD-L1, with the most notable example being miR-200 . miR-200 is a family of miRNAs found in
[51]
two distinct genomic clusters and consists of miR-200a, miR-200b, miR-429, miR-200c, and miR-141 .
miR-200a, miR-200b, and miR-429 form a cluster on chromosome 1, while miR-200c and miR-141 form
a cluster on chromosome 12. miR-200b, miR-200c and miR-429 share the same seed sequence, while
miR-200a and miR-141 have a seed sequence that differs from the others by one nucleotide. The PD-L1
3’-UTR contains one binding site for each seed-sharing functional cluster of miR-200 miRNAs and all
[48]
miR-200a, b, and c have been shown to directly silence PD-L1 expression .

The miR-200 family has been shown to be downregulated in EBV-positive compared to EBV-negative GC
samples, as well as in EBV-negative GC cells following infection with recombinant EBV (rEBV) in vitro [52,53] .
[53]
Whether or how EBV latent proteins downregulate miR-200 remains unclear. Shinozaki et al. reported
that overexpression of any of EBNA1, LMP2A, or BARF0 (a BART transcript) in EBV-negative GC
cells (MKN74 cell line) leads to miR-200a/b transcriptional repression, while overexpression of EBERs
downregulates the mature miRNAs post-transcriptionally. The authors concluded that EBV latent proteins
[52]
and RNAs act synergistically to downregulate miR-200. This is in contrast with a study by Marquitz et al.
on a different EBV-negative GC cell line, AGS. They reported that rEBV-infected AGS (AGS-EBV)
cells showed consistent downregulation in some tumor suppressor miRNAs, including miR-200, when
compared to the parental uninfected cells. However, EBNA1 or LMP1 overexpression in AGS did not affect
[52]
the expression levels of miR-200 and most of the other EBV-downregulated miRNAs . They speculate
that EBV-mediated cellular miRNA downregulation might not be mediated by the latent viral proteins,
[52]
but by viral miRNAs, through their effect on host transcription factors, or by EBERs . Another possible
mechanism of host miRNA downregulation in EBV-infected cells is methylation of their promoter
sequences. EBV infection in GC cells has been shown to promote extensive DNA methylation, partly
through the activity of the latent protein LMP2A, which induces DNA methyltransferases (DNMTs) [54,55] .
Indeed, the miR-200c promoter contains CpG islands that have been shown to be methylated by DNMT3a
[56]
in GC .

Research from our group described an EBV gene/host miRNA/PD-L1 regulation axis in B cell lymphomas,
where the EBV protein EBNA2 induces PD-L1 expression by transcriptionally repressing miR-34a, a
[57]
miRNA that downregulates PD-L1 . EBNA2 is a latency III-associated protein and thus is not expressed
in GC. However, miR-34a has been shown to be downregulated in GC overall and to be transcriptionally
repressed by EBNA1 in EBVaGC [58,59] .

The PD-L1 3’-UTR contains multiple adenosine-uridine (AU)-rich elements (AREs) that are known to
[60]
serve as binding sites for different RBPs . In GC, the RBP tristetraprolin (TTP) has been shown to bind to
[62]
AREs in the PD-L1 3’-UTR and to promote mRNA destabilization, leading to reduced PD-L1 expression .
In addition, in NPC, the EBV latent protein LMP1 reduces TTP expression through extracellular-signal-
[63]
regulated kinase 1/2 (ERK1/2) activation . Nonetheless, there has not been an association between EBV
infection and TTP expression in GC.

Viral miRNAs
EBV expresses 25 precursor miRNAs (pre-miRNAs) and 44 mature miRNAs. Three pre-miRNAs are
derived from the BamHI fragment H rightward open reading frame 1 (BHRF1) region of the viral genome,