Pivotal role of long non-coding ribonucleic acid-X-inactive specific transcript in regulating immune checkpoint programmed death ligand 1 through a shared pathway between miR-194-5p and miR-155-5p in hepatocellular carcinoma

Abstract

Background: Anti-programmed death therapy has thrust immunotherapy into the spotlight. However, such therapy has a modest response in hepatocellular carcinoma (HCC). Epigenetic immunomodulation is a suggestive combinatorial therapy with immune checkpoint blockade. Non-coding ribonucleic acid (ncRNA) driven regulation is a major mechanism of epigenetic modulation. Given the wide range of ncRNAs that co-opt in programmed cell-death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) regulation, and based on the literature, we hypothesized that miR-155-5p, miR-194-5p and long non-coding RNAs (lncRNAs) X-inactive specific transcript (XIST) and MALAT-1 are involved in a regulatory upstream pathway for PD-1/PD-L1. Recently, nutraceutical therapeutics in cancers have received increasing attention. Thus, it is interesting to study the impact of oleuropein on the respective study key players.

Aim: To explore potential upstream regulatory ncRNAs for the immune checkpoint PD-1/PD-L1.

Methods: Bioinformatics tools including microrna.org and lnCeDB software were adopted to detect targeting of miR-155-5p, miR-194-5p and lncRNAs XIST and MALAT-1 to PD-L1 mRNA, respectively. In addition, Diana tool was used to predict targeting of both aforementioned miRNAs to lncRNAs XIST and MALAT-1. HCC and normal tissue samples were collected for scanning of PD-L1, XIST and MALAT-1 expression. To study the interaction among miR-155-5p, miR-194-5p, lncRNAs XIST and MALAT-1, as well as PD-L1 mRNA, a series of transfections of the Huh-7 cell line was carried out.

Results: Bioinformatics software predicted that miR-155-5p and miR-194-5p can target PD-L1, MALAT-1 and XIST. MALAT-1 and XIST were predicted to target PD-L1 mRNA. PD-L1 and XIST were significantly upregulated in 23 HCC biopsies compared to healthy controls; however, MALAT-1 was barely detected. MiR-194 induced expression elevated the expression of PD-L1, XIST and MALAT-1. However, overexpression of miR-155-5p induced the upregulation of PD-L1 and XIST, while it had a negative impact on MALAT-1 expression. Knockdown of XIST did have an impact on PD-L1 expression; however, following knockdown of the negative regulator of X-inactive specific transcript (TSIX), PD-L1 expression was elevated, and abolished MALAT-1 activity. Upon co-transfection of miR-194-5p with siMALAT-1, PD-L1 expression was elevated. Co-transfection of miR-194-5p with siXIST did not have an impact on PD-L1 expression. Upon co-transfection of miR-194 with siTSIX, PD-L1 expression was upregulated. Interestingly, the same PD-L1 expression pattern was observed following miR-155-5p co-transfections. Oleuropein treatment of Huh-7 cells reduced the expression profile of PD-L1, XIST, and miR-155-5p, upregulated the expression of miR-194-5p and had no significant impact on the MALAT-1 expression profile.

Conclusion: This study reported a novel finding revealing that opposing acting miRNAs in HCC, have the same impact on PD-1/PD-L1 immune checkpoint by sharing a common signaling pathway.

Keywords: Hepatocellular carcinoma; Immune checkpoint; MiR-155-5p; MiR-194-5p; Programmed cell-death protein 1/Programmed death ligand 1; X-inactive specific transcript.

Figure 1

Relative expression level of programmed death ligand 1 in liver tissues. Endogenous programmed death ligand 1 expression profile was analyzed in hepatocellular carcinoma patients, cirrhotic and healthy controls using quantified real-time polymerase chain reaction and normalized to B2M as an internal control (housekeeping gene). Screening of programmed death ligand 1 showed that it was enhanced in cirrhotic biopsies (^aP < 0.05) and hepatocellular carcinoma biopsies (^bP < 0.01) compared to healthy controls. HCC: Hepatocellular carcinoma.

Figure 2

Expression profile of lnc-ribonucleic acid X-inactive specific transcript and MALAT-1 in hepatocellular carcinoma tissues. Endogenous X-inactive specific transcript and MALAT-1 lnc-ribonucleic acids expression profile was analyzed in hepatocellular carcinoma (HCC) patients and healthy controls using quantified real-time polymerase chain reaction and normalized to B2M as an endogenous control. A: X-inactive specific transcript lnc-ribonucleic acid showed a significant upregulation in HCC biopsies (P = 0.048); and B: MALAT-1 was significantly down regulated in HCC biopsies (P = 0.043); however, it showed elevated expression in cirrhotic biopsies (P = 0.0136). ^aP < 0.05. HCC: Hepatocellular carcinoma.

Figure 3

Transfection efficiency in Huh-7 cells with miR194-5p and miR-155-5p oligonucleotides. Using quantified real-time-polymerase chain reaction, the transfection efficiency was determined in both mimicked and mock cells 48 h post-transfection for each of the respective mi-ribonucleic acids. MiR-194-5p and miR-155-5p were normalized to RNU6B as an endogenous control. A: Mimicking of miR-194-5p; and B: miR-155-5p resulted in an increase in each of the respective miRNAs, (P = 0.0026) and (P < 0.0001), respectively. The expression levels were compared with the unpaired Student’s t-test. ^bP < 0.01 and ^cP < 0.001.

Figure 4

Impact of miR-194-5p and miR-155-5p on programmed death ligand 1 transcript expression in Huh-7 cells. Following ectopic expression manipulation of (A) miR-194-5p and (B) miR-155-5p in Huh-7 cells, programmed death ligand 1 (PD-L1) transcript expression was assessed using quantified real-time polymerase chain reaction and normalized to B2M as an endogenous control. Mimicking of each of the respective miRNAs resulted in significant upregulation of PD-L1 compared to mock untransfected cells (P = 0.0219) and (P = 0.0209), respectively. On the contrary, PD-L1 transcript expression was significantly downregulated by antagonizing each of the miRNAs in comparison with the mock untransfected cells. ^aP < 0.05.

Figure 5

Impact of miR-155-5p and miR-194-5p on lnc-ribonucleic acids X-inactive specific transcript and MALAT-1 expression in Huh-7 cells. Expression levels of lnc-ribonucleic acids X-inactive specific transcript and MALAT-1 were assessed following transfection of miR-194-5p and miR-155-5p oligomirs using quantified real-time polymerase chain reaction and normalized to the endogenous B2M as a housekeeping gene. A: Ectopic expression of both miR-155 and mir-194 resulted in significant upregulation of X-inactive specific transcript expression, (P = 0.0477) and (P = 0.0026) respectively, compared to mock cells; and B: However, a paradoxical effect of mimicking miR-194-5p and miR-155-5p on MALAT-1 expression profile was observed, as miR-194-5p stimulated the upregulation of MALAT-1 expression (P = 0.0135), whereas mimicking miR-155-5p induced downregulation of MALAT-1 expression in comparison to mock cells (P = 0.0053). ^aP < 0.05 and ^bP < 0.01.

Figure 6

Impact of knockdown of long non-coding ribonucleic acids MALAT-1, X-inactive specific transcript and the negative regulator of X-inactive specific transcript on programmed death ligand 1 expression in Huh-7 cells. Using quantified real-time polymerase chain reaction, the expression profile of programmed death ligand 1 (PD-L1) transcript was determined following transfection of Huh-7 cells with each of the siRNAs for lncRNAs MALAT-1, X-inactive specific transcript and the negative regulator of X-inactive specific transcript. Values were normalized to an endogenous housekeeping gene B2M. PD-L1 expression was induced following knockdown of MALAT-1 (P = 0.0010) compared to mock cells. On the contrary, following down regulation of the negative regulator of X-inactive specific transcript, PD-L1 transcript expression was significantly induced (P = 0.0358) compared to mock cells. PD-L1 expression level was totally unaffected by knocking down MALAT-1 in comparison with mock cells. TSIX: The negative regulator of X-inactive specific transcript.

Figure 7

Net impact of ectopic miR-194-5p and miR-155-5p expression on programmed death ligand 1 expression in the presence of siRNA of lnc-ribonucleic acids X-inactive specific transcript, the negative regulator of X-inactive specific transcript and MALAT-1. The impact of both tumour suppressor miR-194-5p and oncogenic miR-155-5p on programmed death ligand 1 (PD-L1) expression in Huh7 cells in the presence of long non-coding ribonucleic acid X-inactive specific transcript (XIST), the negative regulator of X-inactive specific transcript and MALAT-1 siRNA was similar. Both miRNAs induced upregulation of PD-L1 expression when each was accompanied by MALAT-1 siRNA, while knockdown of the long non-coding ribonucleic acid the negative regulator of X-inactive specific transcript, i.e. upregulation of XIST expression, in the presence of miR-155-5p and miR-194-5p mimics down regulated PD-L1 expression. Ectopic expression of each of the miRNAs with knockdown of XIST had no net impact on PD-L1 expression. ^aP < 0.05 and ^bP < 0.01. TSIX: The negative regulator of X-inactive specific transcript.

Figure 8

Impact of oleuropein on the expression profile of the study key players. The expression pattern of programmed death ligand 1 (PD-L1), micro ribonucleic acids 194-5p and 155-5p as well as long non-coding ribonucleic acids X-inactive specific transcript (XIST) and MALAT-1 were assessed following oleuropein treatment. Values for micro ribonucleic acids were normalized to RNU6B while that for PD-L1, XIST and MALAT-1 were normalized to B2M. Enhanced expression of miR-194-5p was observed following oleuropein treatment (P = 0.0022). However, PD-L1, XIST and miR-155-5p expression pattern were down regulated, (P = 0.0011), (P = 0.0020) and (P = 0.0001), respectively. Oleuropein did not show any significant impact on MALAT-1 expression profile. ^bP < 0.01 and ^cP < 0.001.

Figure 9

Schematic representation of the shared pathway between miR-155-5p and miR-194-5p. This article highlights the novel shared upstream regulatory signaling pathways for programmed cell-death protein 1/programmed death ligand 1 immune checkpoint between paradoxically acting miR-194-5p and miR-155-5p, through lnc X-inactive specific transcript expression modulation. Mimicking of tumor suppressor miR-194-5p as well as oncomiR-155-5p in the Huh-7 cell line showed the same upregulation pattern of X-inactive specific transcript. X-inactive specific transcript was proposed then to be an intermediate player whose upregulation derived the increase in programmed death ligand 1 transcript abundance.