Expression changes of serum LINC00941 and LINC00514 in HBV infection‐related liver diseases and their potential application values

Abstract Background Long non‐coding RNAs (LncRNAs) are considered as potential diagnostic markers for a variety of tumors. Here, we aimed to explore the changes of LINC00941 and LINC00514 expression in hepatitis B virus (HBV) infection‐related liver disease and evaluate their application value in disease diagnosis. Methods Serum levels of LINC00941 and LINC00514 were detected by qRT‐PCR. Potential diagnostic values were evaluated by receiver operating characteristic curve (ROC) analysis. Results Serum LINC00941 and LINC00514 levels were elevated in patients with chronic hepatitis B (CHB), liver cirrhosis (LC), and hepatocellular carcinoma (HCC) compared with controls. When distinguishing HCC from controls, serum LINC00941 and LINC00514 had diagnostic parameters of an AUC of 0.919 and 0.808, sensitivity of 85% and 90%, and specificity of 86.67% and 56.67%, which were higher than parameters for alpha fetal protein (AFP) (all p < 0.0001). When distinguishing HCC from LC, CHB, or LC from controls, the combined detection of LINC00941 or LINC00514 can significantly improve the accuracy of AFP test alone (all p < 0.05). Conclusions LINC00941 and LINC00514 were increased in the serum of HBV infection‐associated liver diseases and might be independent markers for the detection of liver diseases.

liver cancers, and ranks fourth and third in incidence and mortality among all malignant tumors; in addition, compared with females, males have a higher incidence and poorer prognosis. 3,4 HCC is mainly caused by hepatitis virus infection, alcohol abuse, non-alcoholic steatohepatitis, toxin exposure, and metabolic syndrome. 5 Chronic infection of hepatitis B virus (HBV) and aflatoxin exposure are the main pathogenic factors of HCC in China, and HCC caused by chronic HBV infection accounts for more than 80% of all HCC. 6 In addition to liver ultrasound, serum alpha fetal protein (AFP) detection is the main method for extensive screening of HCC in China. However, due to its low sensitivity and specificity, the detection rate of HCC is not high, so that many HCC patients miss the optimal surgical period. Therefore, it is very important to explore novel and effective serum markers to improve the detection rate and prognosis of HCC.
Non-coding RNA is currently a research hotspot in the field of is increased in pancreatic cancer, colorectal cancer, lung cancer, etc. [7][8][9][10] It can promote tumor progression through a variety of signaling pathways, such as proliferation, metastasis, invasion, and so on. [7][8][9]11 It can also be used as a diagnostic or prognostic marker for gastric cancer, lung cancer, head and neck squamous cell carcinoma, and other tumors [12][13][14] and could predict the recurrence-free survival and overall survival of HCC. 15 On the other hand, it has been reported that LINC00514 is highly expressed in the tissues and cells of breast and pancreatic cancer and can promote tumor occurrence and development by regulating related microRNAs. 16,17 However, so far, no studies have reported their diagnostic value in HCC.
In this research, we detected the expression of LINC00941 and LINC00514 in healthy controls and patients with HBV infectionrelated liver disease and assessed its correlation with basic characteristics of patients with HCC. The diagnostic values of LINC00941 and LINC00514 in liver diseases were also analyzed by receiver operating characteristic curve (ROC).

| Study population
All subjects were collected from inpatient department of Renmin

| Sample collection
All vacuum blood collection tubes were purchased from BD Company. The yellow head tube to promote blood coagulation and the pearl-white head tube to promote blood coagulation of all subjects were collected on an empty stomach for more than 8 h in the morning. The yellow head tube blood was used for routine biochemical indexes, AFP, LINC00941, and LINC00514 detection, and the pearl-white head procoagulant blood was used for HBV DNA detection. After collection, the blood was placed at room temperature for 15min. When the blood was completely coagulated, the serum was separated after centrifugated at 3500 r/min for 5 min for use.

| Laboratory analysis
Automatic biochemical analyzer (Siemens, Germany) and supporting analysis reagents were used to detect serum alanine ami-

| Serum LINC00941 and LINC00514 extraction and qRT-PCR analysis
The serum LINC00941 and LINC00514 were extracted with Beijing Biotech (blood RNA extraction and adsorption column type) kit.
The above two non-coding RNAs were quantified by fluorescence quantitative PCR using the reverse transcription and quantitative PCR kits (RR036A and RR091A) of Takara, Japan, and GAPDH as the housekeeping gene. Reverse transcription was performed at 37℃ Tamhane'ST2 test was used for non-homogeneity. The quantitative data that did not obey the normal distribution was represented by the median and interquartile ranges, and the Mann-Whitney test was used to analyze the corresponding data. Pearson's correlation was used to evaluate the correlation among all indicators. ROC curve was used to analyze the diagnostic value of LINC00941, LINC00514, and AFP for HCC. All data were analyzed by two-sided test, and p < 0.05 was considered as statistically significant.

| Characteristics of healthy controls and patients with HBV infection-related liver disease
The main characteristics of all the study population are summarized in Table 1. First, there was no statistically significant difference in age between the groups. Regarding the liver biochemical indicators ALT, AST, ALP, GGT, ALB, TBIL, DBIL, the differences between the four groups were statistically significant (all p < 0.0001). For the level of HBV DNA, the CHB group was significantly higher than the LC group and the HCC group (all p < 0.0001). In the analysis of serum tumor markers, result showed that the AFP level of HCC patients was obviously increased than that of the control group, CHB and LC groups, and the difference was statistically significant.

| Comparison of serum LINC00941 and LINC00514 levels in controls, CHB, LC and HCC
Furthermore, we used the LSD test to evaluate the serum levels of LINC00941 and LINC00514 in the controls, CHB, LC and HCC groups. As shown in Figure 1A, serum LINC00941 levels in the CHB, LC and HCC groups were significantly higher than those in the control group (all p < 0.0001), and compared with the patients with LC, the level of serum LINC00941 in CHB and HCC groups were obviously decreased (all p < 0.0001). Compared to the controls, the serum LINC00514 level was significantly increased in patients with CHB, LC and HCC (all p < 0.0001); and the serum LINC00514 level was significantly lower in the CHB group and the HCC group than that of LC group (all p < 0.0001, Figure 1B).

| Relationship between serum LINC00941 and LINC00514 expression and basic biochemical indexes in patients with HCC
To further evaluate whether serum LINC00941 and LINC00514 expression are related to the liver function, HBV viral load and AFP level of HCC patients, We analyzed the correlation between the high and low expression of two lncRNAs and the above indexes. The results showed that serum LINC00941 and LINC00514 had no correlation with liver function index, HBV viral load and AFP ( Table 2).

| Serum levels of LINC00941, LINC00514 and AFP at different HCC stages and liver function grades
To assess the changes of LINC00941, LINC00514, and AFP levels in the progression of HCC, patients with HCC were divided into early, middle and advanced stages, according to the Barcelona Clinic Liver Cancer (BCLC). We found that serum level of AFP in advancedstage HCC was significantly higher than early stage and middle stage (all p < 0.01, Figure 2A). However, the levels of LINC00941 and LINC00514 showed no difference in different stages of HCC but showed a gradual downward trend ( Figure 2B,C). According to Child-Pugh class, patients with HCC were divided into class A, B, and C. The results indicated that the AFP level of class C patients was obviously higher than that of class A patients, while the LINC00941 and LINC00514 levels showed no difference in different liver function grades, and LINC00514 showed a trend of progressive decrease in different grades ( Figure 2D-F).

| Diagnostic value of serum LINC00941, LINC00514 and AFP
To explore whether serum LINC00941 and LINC005141 can be LINC00941 and LINC00514 showed no significant advantage in distinguishing HCC from CHB compared to AFP ( Figure 3B). When distinguishing HCC from LC, LINC00941 and LINC00514 combined with AFP significantly improved the accuracy of HCC diagnosis (0.820 vs. 0.668 and 0.835 vs. 0.668, all p < 0.01, Figure 3C). Compared with AFP alone, LINC00941 and LINC00514 alone or in combination with AFP improved sensitivity and accuracy in the diagnosis of CHB and LC (used healthy controls as control group, Figure 3D,F). When differentiating LC and CHB, LINC00941 alone or in combination with AFP significantly improved the sensitivity and accuracy of LC diagnosis compared with AFP alone (all p < 0.01, Figure 3E) ( Table 3).

| DISCUSS ION
The prevalence of liver cancer is related to its many pathogenic factors and complex pathogenesis, which also means that it is difficult to diagnose with a single biomarker. Therefore, the combined mechanistic studies have shown that the LINC01134/SP1/p62 axis modulated OXA resistance by changing cell viability, apoptosis and mitochondrial homeostasis in vitro and in vivo, suggesting that the LINC01134/SP1/p62 axis may be a promising strategy to overcome OXA chemotherapy resistance. 23 These findings suggest that ln-cRNAs play an important role in the occurrence and development, treatment, and prognosis monitoring of HCC.
In our study, we detected the expression levels of LINC00941 and LINC00514 in the serum of controls, CHB, LC, and HCC patients, and found that compared with the healthy control group, the serum levels of LINC00941 and LINC00514 in the CHB, LC and HCC groups were significantly increased. The levels of LINC00941 and LINC00514 in the LC group were significantly higher than those in the CHB and HCC groups.

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest.

AUTH O R CO NTR I B UTI O N
PZ, JC, and DT proposed the concept of the work. JC carried out most of the experimental work and wrote the paper. HL provided critical reviews in order to promote the manuscript. All authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.