lncRNA XIST Interacts with Regulatory T Cells within the Tumor Microenvironment in Chronic Hepatitis B-Associated Hepatocellular Carcinoma

Objective: Alterations in the expression of several long non-coding RNAs (lncRNAs) have been shown in chronic hepatitis B-associated hepatocellular carcinoma (CHB-HCC). Here, we aimed to investigate the association between the expression of inflammation-associated lncRNA X-inactive specific transcript (XIST) and the type of inflammatory cells within the tumor microenvironment. Material and Methods: Twenty-one consecutive cirrhotic patients with CHB-HCC were included. XIST expression levels were investigated on formalin-fixed paraffin-embedded (FFPE) tumoral and peritumoral tissue samples by real-time polymerase chain reaction (RT-PCR). Immunohistochemical staining for CD3, CD4, CD8, CD25, CD163, CTLA4, and PD-1 were performed. The findings were statistically analyzed. Results: Of the 21 cases, 11 (52.4%) had tumoral and 10 (47.6%) had peritumoral XIST expression. No significant association was found between the degree of inflammation and XIST expression. The number of intratumoral CD3, CD4, CD8 and CD20 positive cells was higher in XIST-expressing tumors, albeit without statistical significance. Tumoral and peritumoral XIST expression tended to be more common in patients with tumoral and peritumoral CD4high inflammation. The number of intratumoral CD25 positive cells was significantly higher in XIST-expressing tumors (p=0.01). Tumoral XIST expression was significantly more common in intratumoral CD25high cases (p=0.04). Peritumoral XIST expression was also more common among patients with CD25high peritumoral inflammation, albeit without statistical significance (p=0.19). Conclusion: lncRNA XIST is expressed in CHB-HCC and its expression is significantly associated with the inflammatory tumor microenvironment, particularly with the presence and number of CD25 (+) regulatory T cells. In vitro studies are needed to explore the detailed mechanism.

We recently investigated inflammation-associated lncRNA expression in formalin-fixed paraffin-embedded (FFPE) tissue samples (chronic viral hepatitis (CVH)-associated HCC, peritumoral cirrhotic parenchyma, nontumoral cirrhotic CVH parenchyma, and normal liver) using a realtime polymerase chain reaction (RT-PCR) array panel of 84 inflammation-associated lncRNAs, and then, based on the RT-PCR array results, we performed RT-PCR assay testing to compare expression patterns of 7 inflammationassociated lncRNAs between CVH-HCC, peritumoral cirrhotic parenchyma, and nontumoral CVH samples as a second step (manuscript in preparation).One of the studied lncRNAs was lncRNA X-inactive specific transcript (XIST), which we found to be associated with the presence of negative prognosticators in CVH-HCC (manuscript in preparation).XIST is expressed in all female somatic cells, especially during the early phases of development, and in germ cells in males but its expression in neoplastic and nonneoplastic diseases is variable and is considered to result from abnormal activation of X chromosome-related genes during pathological processes (14)(15)(16).
In addition to its significant association with negative prognosticators, we also observed that peritumoral XIST expression was related with neutrophil predominant inflammation in peritumoral cirrhotic parenchyma.Thus, we aimed to investigate the association between the expression of inflammation-associated lncRNA XIST and the type of inflammatory cells within the tumor microenvironment, i.e., within the HCC focus and nearby cirrhotic parenchyma, in the current study.

Case Selection and Collection of Clinicopathological Data
The study was approved by the institutional ethics committee (2019/05-70).A total of 21 consecutive cirrhotic patients with CHB-HCC who had underwent liver transplantation without prior interventional treatment were included.Clinicopathological data were retrieved from the hospital records.Hematoxylin-eosin-stained slides of all cases were re-evaluated, and the degree and type of inflammation in neoplastic and peritumoral non-neoplastic tissue were noted.
One high power field containing the highest number of positive cells were photographed, and the number of the positive cells were counted.The median was determined for each group.In addition to negative/positive categorization, values less than the median value was grouped as "low", and values more than the median value were grouped as "high" for each marker except for CTLA4 (Figure 1).CTLA4 staining was grouped as "negative" or "positive".

Measurement of XIST Expression
XIST primary assay (RT² QPCR Primer Assay, Qiagen, Germany) was used to detect XIST expression levels on FFPE tissue samples by RT-PCR.Hematoxylin-eosin-stained slides were reviewed to select representative tumor and peritumoral cirrhotic parenchyma (within 1 cm distance from the HCC focus) areas.Selected areas were marked.Then, five sections of 7 microns from the selected areas were taken and placed into Eppendorf tubes.RNA extraction was done according to manufacturer's recommendations using a kit for FFPE tissues (RNEASY FFPE Kit, Qiagen, Germany).cDNA synthesis and preamplification were then done using a suitable kit (RT² PREAMP cDNA Synthesıs Kit, Qiagen, Germany) before preparation for Figure 1: Immunohistochemical evaluation.The number of the positive cells was counted in one high power field and the median was determined.In addition to negative/positive categorization, values less than the median value were grouped as "low", and values more than the median value were grouped as "high" for each marker except CTLA-4, which was evaluated either as negative or positive.A) Intratumoral CD25negative, B) Intratumoral CD25low, and C) Intratumoral CD25high.

A B C
PCR.RT2 SYBR Green Mastermix (Qiagen, Germany) was used during sample preparation.Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the reference gene.The Ct value was determined for each case and the expression status was interpreted qualitatively, i.e., as expressed or not-expressed.

Statistical Analysis
Statistical analyses were performed using SPSS ver.24 (IBM, USA).In addition to frequency analyses, the chisquare test was used to compare categorical variables and Kruskal-Wallis's test was used for comparisons between 2 or more groups.The association between the parameters was sought using the Pearson or Spearman correlation test.p<0.05 was considered statistically significant.

Clinicopathological Features
All cases had histopathologically verified cirrhosis.Two cases were female (F:M = 0.10

Immunophenotype of the Tumor Microenvironment
The number of the CD3, CD4, CD8 and CD20 positive cells were significantly lower in the tumor compared to the peritumoral cirrhotic parenchyma (p<0.05;Table II).There was moderate correlation between intratumoral and peritumoral CD25 positive cell counts (r=0.57;p=0.008,Pearson correlation test).The intratumoral CD25 positive cell count was also significantly correlated with intratumoral CD3, CD4, CD8, CD20, and CTLA4 positive cell counts (p<0.05).
The tumoral CD8 score was significantly associated with the peritumoral CD3 score (p=0.028).However, no other significant difference or association was found between the tumoral and peritumoral groups regarding the distribution of the inflammatory cell subgroups (p>0.05).

Association Between XIST Expression and the Immunophenotype of the Inflammatory Cells
The number of intratumoral CD3, CD4, CD8, and CD20 positive cells was higher in XIST-expressing tumors but the difference did not reach statistical significance (Table III The number of intratumoral CD25 positive cells was significantly higher in XIST-expressing tumors (p=0.01)(Table III) (Figure 1).Peritumoral XIST expression was also more common among patients with CD25high peritumoral inflammation, albeit without statistical significance (5/8 vs.4/12; p=0.19), and no significant correlation was found between peritumoral XIST expression level and peritumoral CD25 count (p=0.09).
There was a moderate negative correlation between peritumoral XIST Ct values and peritumoral CTLA-4 positive cell counts (r=-67; p=0.031,Pearson test), although there was no significant correlation between the peritumoral XIST expression level and the peritumoral CTLA4 score (p>0.05).

DISCUSSION
In this study, we evaluated the association between the expression of lncRNA XIST and the type of inflamma-  (25,27).These different findings may simply reflect the fact that the effect of lncRNAs is generally dependent of other factors such as microRNA expression.In any case, we believe that the association between XIST expression and neutrophilic infiltration deserves further investigation.
The number of the CD3 positive cells was significantly lower in the tumor compared to peritumoral cirrhotic parenchyma.However, curiously, we found a significant association between peritumoral XIST expression and tumoral CD3 positive cell count.In addition, the number of the intratumoral CD3, CD4, CD8 and CD20 positive cells were higher in XIST expressing tumors, although no statistical significance could be reached.These findings indicate that XIST expression induces inflammation, particularly T cell proliferation, in the tumor microenvironment, most likely due to stimulation of cytokine production as previously demonstrated (28,29).
We also observed that tumoral and peritumoral XIST expression were more common in patients with CD4high inflammation, albeit without statistical significance, probably due to the small number of the patients in the study tory cells within the tumor and peritumoral cirrhotic liver parenchyma.
Our study is one of the few studies on XIST expression in hepatocellular carcinoma that was conducted using tissue samples (17)(18)(19)(20)(21)(22).Overall, about half of the cases had tumoral and/or peritumoral XIST expression.Tumoral and peritumoral XIST Ct values were strongly correlated.Although our study group is not appropriate for gender comparison regarding XIST expression considering there were only 2 women, 9/11 cases showing tumoral expression were male, and considering XIST is not normally expressed in somatic cells in males (14)(15)(16), its expression in CHB-HCC and peritumoral liver tissues is considered pathological.
XIST is a critical regulator in X chromosome inactivation (hence the name), but it has also been shown to be involved in many biological and pathological processes, including cancer, inflammation, and cardiac and neurological diseases (23,24).XIST has been demonstrated to alter the inflammatory response via the NF-κB pathway, which is also an important pathway in development of inflammation-associated HCC (5,25,26).All 4 patients with neutrophil predominant peritumoral inflammation expressed XIST in the peritumoral parenchyma in the current study.
There is limited data on the association between XIST expression and neutrophilic inflammation in the literature.group.However, this finding seems to be consistent with the literature.Recently, high-expression of XIST in CD4positive cells and natural killer cells has been suggested to trigger the proliferation of naïve CD4 (+) T cells in primary biliary cholangitis (30).In another study, high XIST expression has been found to be associated with the CD4-positive T cell level in systemic lupus erythematosus patients (31).
CD4+CD25+ regulatory T cells are suppressor T cells and the presence of IL-2 is required for their survival (32)(33)(34).Similar to our study, CD4+CD25+ regulatory T cells have been shown to increase in CHB and CHB-HCC, and they are thought to inhibit the antitumoral immune response, resulting in a tumor promoting environment (35)(36)(37)(38)(39).In addition, one of the major findings in the current study was the significant association between the tumoral XIST expression and the number of the intratumoral CD25positive inflammatory cells.XIST was more frequently expressed in intratumoral CD25high cases.Peritumoral XIST expression also tended to be more frequent in patients with CD25high peritumoral inflammation, albeit without statistical significance.These findings suggest that increase in CD25-positive T cells may be the result of XIST expression.Activated mature T cells have been shown to restore XIST expression, and the XIST location pattern of regulatory T cells has been demonstrated to differ dependent of the stimulation time in mice (40).There is also evidence of XIST regulating the proliferation of T helper subsets (41,42).Therefore, there may be an interaction between XIST-expressing hepatocytes and T cells within the CHB-HCC microenvironment, causing the activation of CD25positive regulatory T cells.Moreover, NF-κB has been claimed to control IL-2 expression during T cell development (43).Considering that XIST acts via the NF-κB pathway (see above), it may be facilitating IL-2 expression, and thus CD25 expression in T cells.However, in vitro studies are required to demonstrate the possible direct association between XIST expression and CD25 positive T cells.
The most important limitation of our study was the small number of patients.However, we believe that this study provides essential findings for possible future in vitro studies on the association between XIST expression and regulatory T cells.
In conclusion, lncRNA XIST is expressed in CHB-HCC and induces inflammation, and its expression is significantly associated with the inflammatory tumor microenvironment, particularly with CD25 (+) regulatory T cells.
In vitro studies are needed to establish the detailed mechanism and contributing pathways of this association.

Table I :
Clinicopathological characteristics.

Table II :
Comparison of immunohistochemical cell counts between the tumor and peritumoral parenchyma samples.
While Shenoda et al. have claimed that XIST weakens the acute inflammatory response in female cells, XIST silencing has been demonstrated to reduce neutrophil extracellular trap formation in another recent study

Table III :
Association between the phenotype of the inflammatory cells and XIST expression.