Hepatitis B virus (HBV) infection is a major risk factor for the development of hepatic cirrhosis (HC) and hepatocellular carcinoma (HCC), which are associated with very high morbidity and mortality rates worldwide. Many studies have shown that long noncoding RNAs (lncRNAs) that are highly expressed in HCC (lncRNA-HEIH) and highly upregulated in liver cancer (lncRNA-HULC) have been implicated in the development and progression of hepatitis B-related HC and HCC. In this study, reverse transcription and quantitative PCR were used to detect the expression of lncRNA-HEIH and lncRNA-HULC and western blot analysis to detect the expression of hepatitis B X-interacting protein (HBXIP). RNA immunoprecipitation was used to detect the interaction of HBXIP with lncRNA-HULC and lncRNA-HEIH. The results showed that lncRNA-HEIH, lncRNA-HULC, and HBXIP were upregulated in hepatitis B patients, particularly those with hepatitis B-related HCC. Both lncRNA-HEIH and lncRNA-HULC interacted with HBXIP. These results suggest that lncRNA-HEIH and lncRNA-HULC interact with HBXIP in hepatitis B-related diseases.
Hepatitis B virus (HBV) infection remains a major public health concern, affecting more than 350 million people worldwide, despite the advent of effective vaccines and other control measures [
Long noncoding RNAs (lncRNAs), a class of RNA segments >200 nucleotides in length, have been considered to be the “noise” of genome transcription owing to their limited protein-coding capacity [
The aims of this study were to quantify the expression levels of lncRNA-HULC, lncRNA-HEIH, and HBXIP in patients with HBV infection and hepatitis B-related diseases.
The study protocol was approved by the Ethics Committee of People’s Hospital of Anji and conducted in accordance with the tenets of the Declaration of Helsinki and the ethical guidelines for medical and health research involving human subjects as established by the National Institutes of Health and the Committee on Human Research of People’s Hospital of Anji. Written informed consent was obtained from all patients or their lineal relatives for the use of peripheral blood and liver tissues.
Peripheral blood samples were collected from 75 HBV-positive patients and 25 HBV-negative normal controls who received treatment or medical examinations at People’s Hospital of Anji. Patients with infections of HBV or any other virus (e.g., hepatitis virus A, C, D, E, and I) or liver diseases (e.g., hepatic cyst and hepatic metastasis) were excluded from analysis. The 100 study participants were allotted to one of the four following groups (
Clinical features of patients.
Parameter | Control | HBV | HBV + HC | HBV + HCC | |
---|---|---|---|---|---|
Gender | |||||
Female | 13 | 14 | 12 | 10 | >0.05 |
Male | 12 | 11 | 13 | 15 | |
HBsAg | |||||
Negative | 25 | 0 | 0 | 0 | <0.05 |
Positive | 0 | 25 | 25 | 25 | |
Age | 45.07 ± 15.06 | 40.18 ± 16.25 | 42.31 ± 8.56 | 45.62 ± 11.28 | >0.05 |
ALP (U/L) | 55.58 ± 12.01 | 91.46 ± 12.96 | 121.77 ± 16.89 | 157.29 ± 21.78 | <0.05 |
ALB (g/L) | 48.44 ± 8.88 | 43.32 ± 7.58 | 28.21 ± 6.69 | 30.37 ± 6.84 | <0.05 |
TBIL ( |
15.23 ± 3.67 | 22.24 ± 4.52 | 33.52 ± 7.17 | 40.44 ± 8.03 | <0.05 |
DBIL ( |
4.28 ± 1.13 | 8.44 ± 1.61 | 17.22 ± 3.35 | 27.24 ± 5.98 | <0.05 |
AFP ( |
4.95 ± 1.13 | 26.83 ± 4.67 | 52.79 ± 7.76 | 122.74 ± 18.76 | <0.05 |
ALT (U/L) | 25.48 ± 6.68 | 181.67 ± 21.90 | 69.28 ± 11.11 | 78.24 ± 10.94 | <0.05 |
AST (U/L) | 22.14 ± 5.11 | 196.75 ± 20.29 | 89.89 ± 13.22 | 102.25 ± 14.11 | <0.05 |
HBV-DNA (copies/mL) | 0 | (7.12 ± 0.28) × 105 | (2.23 ± 0.47) × 104 | (7.35 ± 0.55) × 104 | <0.05 |
HBsAg: hepatitis B surface antigen; ALP: alkaline phosphatase; ALB: albumin; TBIl: total bilirubin; DBIl: direct bilirubin; AFP: alpha-fetoprotein; ALT: alanine aminotransferase; AST: aspartate aminotransferase.
HBV-positive HCC tissues and corresponding adjacent noncancerous liver tissues (NT) were obtained from patients in the HBV + HCC, HBV-HCC, HBV + NT, and HBV-NT groups who underwent resection in our hospital. On account of the ethical limitation, liver tissues were not collected from the normal controls, HBV carriers, or HBV-positive patients with HC. Thus, normal and cirrhotic liver tissues were not assessed in this study. The clinicopathological characteristics of the study participants are summarized in Table
Clinical features of patients.
Parameter | HBV + HCC | HBV-HCC | |
---|---|---|---|
Gender | |||
Female | 10 | 12 | >0.05 |
Male | 15 | 13 | |
HBsAg | |||
Negative | 0 | 25 | <0.05 |
Positive | 25 | 0 | |
Age | 45.62 ± 11.28 | 44.71 ± 9.98 | >0.05 |
ALP (U/L) | 166.29 ± 36.12 | 150.49 ± 29.98 | >0.05 |
ALB (g/L) | 34.56 ± 10.18 | 33.33 ± 17.24 | >0.05 |
TBIL ( |
42.49 ± 10.36 | 45.05 ± 6.21 | >0.05 |
DBIL ( |
31.75 ± 4.98 | 30.23 ± 5.69 | >0.05 |
AFP ( |
125.78 ± 19.27 | 126.09 ± 22.08 | >0.05 |
ALT (U/L) | 90.57 ± 12.14 | 91.07 ± 25.07 | >0.05 |
AST (U/L) | 111.15 ± 9.72 | 126.77 ± 25.69 | >0.05 |
HBV-DNA (copies/mL) | (8.21 ± 0.69) × 104 | 0 | <0.05 |
HBsAg: hepatitis B surface antigen; ALP: alkaline phosphatase; ALB: albumin; TBIl: total bilirubin; DBIl: direct bilirubin; AFP: alpha-fetoprotein; ALT: alanine aminotransferase; AST: aspartate aminotransferase.
RT-qPCR was used to detect the expression of lncRNA-HULC and lncRNA-HEIH. Total RNA was isolated from the peripheral blood samples, liver tissues, and HepG2.2.15 cells using TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA) and then reverse transcribed into cDNA with the Veriti 96-Well Thermal Cycler (Applied Biosystems, Carlsbad, CA, USA) using the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa Biotechnology (Dalian) Co. Ltd., Dalian, China). Each RT-qPCR reaction contained 2
RIP analyses were performed to detect the interaction between LncRNA-HEIH/LncRNA-HULC and HBXIP using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (EMD Millipore Corporation, Billerica, MA, USA) in accordance with the manufacturer’s instructions. Briefly, liver tissues or harvested cells were lysed in RIP lysis buffer and then incubated with RIP buffer containing magnetic beads conjugated to antibodies (Abs) against HBXIP (Abcam, Cambridge, UK) or normal rabbit immunoglobulin (Ig) G (Abcam). The samples were incubated with proteinase K to digest the proteins, then the coprecipitated RNA was detected by RT-qPCR.
Total protein was extracted from the liver tissues and HepG2.2.15 cells, then quantified, diluted with 5× loading buffer to the same concentration, denatured at 95°C, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene fluoride membranes, which were blocked with 5% skimmed milk at room temperature for 2 h and then incubated with Abs against HBXIP (dilution, 1 : 1000; Abcam) and GAPDH (dilution, 1 : 3000; Abcam) overnight at 4°C. Afterward, the membranes were washed three times with Tris-buffered saline with Tween 20, incubated with horseradish peroxidase-conjugated goat anti-rabbit Ab (dilution, 1 : 4000; Abcam), and washed three times with Tris-buffered saline with Tween 20. Then, the protein bands were visualized using an enhanced chemiluminescence reagent (Santa Cruz Biotechnology Inc., Dallas, TX, USA) and quantified by densitometric scanning with the Fusion-FX7 system (Vilber Lourmat, Collégien, France). The mean optical density of the samples was normalized to that of GAPDH.
All data are expressed as the mean ± standard deviation and were analyzed using the
According to the RT-qPCR results, the expression levels of LncRNA-HEIH and LncRNA-HULC were significantly upregulated in the peripheral blood of HBV-positive patients (HBV, HBV + HC, and HBV + HCC groups), as compared to the control group (
Expression patterns of LncRNA-HEIH and LncRNA-HULC in peripheral blood and liver tissues. (a) Expression levels of LncRNA-HEIH and LncRNA-HULC in peripheral blood (
RIP assays were performed to determine whether HBXIP interacts with LncRNA-HEIH and LncRNA-HULC. RNA obtained from the RIP assay using Abs against HBXIP and IgG was subjected to RT-qPCR analysis. The results indicated that the expression levels of LncRNA-HEIH and LncRNA-HULC were greater in the samples pretreated with HBXIP Ab as compared to the those pretreated with IgG (
LncRNA-HEIH and LncRNA-HULC coimmunoprecipitates with HBXIP. (a) The expression levels of LncRNA-HEIH and LncRNA-HULC in the samples co-precipitated by HBXIP (
With the development of high-resolution microarrays and massively parallel sequencing technology, it is widely believed that more than 90% of the human genome is actively transcribed into noncoding RNAs (ncRNAs) and less than 2% actually encodes proteins [
The expression levels of lncRNA-HULC and lncRNA-HEIH are relatively high in HCC. LncRNA-HULC is located on chromosome 6p24.3 and regarded as the first lncRNA with highly specific upregulation in HCC, suggesting that it can be used as a promising noninvasive novel biomarker for the diagnosis and/or prognosis of HCC [
In summary, the present findings demonstrate that the interactions of lncRNA-HULC and lncRNA-HEIH with HBXIP might be involved in the occurrence of hepatitis B-related diseases. Understanding the relationship of lncRNA-HULC and lncRNA-HEIH with HBXIP in hepatitis B and hepatitis B-related diseases may lead to the development of novel therapeutic interventions to ameliorate hepatitis network dysfunction and associated morbidities.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors have no conflict of interests to declare.
Lingjuan Ruan and Lifei Huang contributed equally to this work.
We thank the patients and families for the donation of peripheral blood and liver tissues. This work was supported by Public Welfare Application Research Project of Huzhou Science and Technology Bureau, China (Project no: 2017GY67).