Inhibition of miR‐148a‐3p resists hepatocellular carcinoma progress of hepatitis C virus infection through suppressing c‐Jun and MAPK pathway

Abstract Objectives The present study was committed to investigate the role of miR‐148a‐3p in HCC infected with hepatitis C virus (HCV) and the regulatory mechanism of miR‐148a‐3p/c‐Jun/MAPK signalling pathway. Methods Differential analysis and GSEA analysis were performed with R packages. QRT‐PCR and Western blot were used to detect RNA or protein level, respectively. The targeted relationship between miR‐148a‐3p and c‐Jun was predicted by TargetScan database and determined by double luciferase reporter assay. MTT assay and flow cytometry were used to evaluate cell proliferation, cell cycle and cell apoptosis, respectively. Results C ‐Jun was up‐regulated, and MAPK signalling pathway was activated in HCV‐infected HCC cells. C‐Jun expression regulated inflammation‐related gene expression and had an influence on cell proliferation, cell cycle and cell apoptosis. MiR‐148a‐3p, down‐regulated in HCV‐infected HCC cells, could target c‐Jun mRNA to suppress c‐Jun protein expression. Conclusions MiR‐148a‐3p suppressed the proliferation of HCC cells infected with HCV through targeting c‐Jun mRNA.

is considered as HCV envelope protein, has aroused the interests in the therapy of HCV. Zhao et al revealed that E2 could stimulate the proliferation of human hepatoma cell via the MAPK/ERK signalling pathway in 2005. 5 Results of Liu et al displayed that synthesized peptides 705-734 from E2 could induce the maturation through p38 MAPK signalling. 1 The recent method of HCV therapy is direct-acting antivirals (DAAs). 6 However, uncertainty about the optimal timing of DAA therapy and other untoward response make more developed and efficient therapy of HCV urgent.
Mitogen-activated protein kinase (MAPK) family, a group of serine/threonine kinases, plays an essential role in the regulation of the cell activities, for instance, apoptosis, cell cycle and transcription. It could be divided into three group-p38 family kinases, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). 3,5,7 Many researches have been implemented to uncover the specific mechanism of MAPK. Besides the studies mentioned above in HCV, MAPK signalling pathway could also be carried out in other fields. 8,9 For example, data in the study of Li et al showed that Angiotensin II regulated the expression of miR-143/145 through p38 MAPK signalling pathway in the pathogenesis of aortic dissection. 9 However, the mechanisms of MAPK signalling pathway remain to be explored and supplemented.
C-Jun, a member of the activating protein 1 (AP-1) transcription factor family, has been proved to have a significant role in cellular processes, such as apoptosis, migration and differentiation. 10 Mariani demonstrated that Jun oncogene was high expressed in high aggressive sarcomas and could block adipocytic differentiation. 11 Zenz revealed that c-Jun had a regulation on the development of eyelid closure and skin tumour via EGFR signalling. 12 Results of Liu et al displayed the function of HP1a/KDM4A in the mechanism of c-Jun regulation. 10 MiRNA is a length of 19-25 nt (nucleotides) noncoding RNAs. 13 MiRNAs have a regulation in cell proliferation, differentiation, apoptosis and other cellular processes via enhancing or inhibiting the expression of many mRNAs. [14][15][16] Numerous previous studies have manifested that miR-148a-3p was involved in laryngeal squamous cell carcinoma (LSCC), bladder cancer, gastric cancer and other several cancers. [13][14][15] However, there is a gap in the study of miR-148a-3p in the field of HCV, especially related to miRNA.
In the current study, we found that miR-148a-3p was expressed remarkably low and MAPK signalling pathway was activated through exploring the influences of HCV on the infection cells. Further, we tried to confirm the regulation of miR-148a-3p on HCV infection and the internal mechanism.

| Patients
Healthy individuals (normal, n = 15), hepatitis patients infected with hepatitis C virus (HCV, n = 15) and HCC patients infected with HCV (HCC, n = 15) were recruited from the Affiliated Hospital of Youjiang Medical College for Nationalities in this study. The patients' clinical characteristics are provided in Table S1. All patients and healthy individuals obtained their written informed consent, and this study protocol was approved by the Affiliated Hospital of Youjiang Medical College for Nationalities. Blood was collected from the cubital vein with anticoagulant (heparin sodium) and processed immediately.

| Microarray analysis
The expression profile GSE44210 (GPL6480) was derived from gene expression omnibus (GEO) database to analyse differentially

| Gene set enrichment analysis (GSEA)
The enrichment analysis for KEGG pathway was performed with the normalized mRNA expression profiles of GSE44210 by GSEA v3.0 software. The enriched pathways were visualized by dotplot and gesaplot with ggplot2, grid, devtools and easygplot2 packages.

| MTT assay
Cell viability was quantified by MTT assay. After transfecting with c-Jun siRNAs or miR-148a-3p mimics or inhibitor 24 hours, cells were resuspended and then 2000 cells were seeded in 96-well plates. At 0 hour, 12 hours, 24 hours, 36 hours and 48 hours, cells were treated with MTT reagent (100 μL of fresh serum-free medium with 0.5 g/L of MTT) at 37°C for 4 hours, following by adding with 50 μL DMSO each well for 10 minutes. The absorbance of each well was measured by microplate photometer at 450 nm.

| Double luciferase reporter assay
Through the TargetScan database, it was predicted that the c-Jun mRNA 3′UTR had seed sequences which were partially complementary to miR-148a-3p. C-Jun 3′UTR fragments and their mutants were cloned the downstream of the luciferase coding region in the pGL3-

| HCV up-regulated c-Jun and activated MAPK pathway in Huh7 cells
Limma packages were applied to significance analysis of differentially expressed genes (DEGs) between JFH1-infected and uninfected Huh-7.5.1 cell from GSE44210. The criteria were set as follows for screening differential genes in two groups: P < 0.05 and the absolute value of logFC >2. The cluster analysis was performed, and the heat map was drawn according to their expression levels. As shown in Figure 1A, C-Jun was up-regulated in HCV infection group. Using GSEA for KEGG pathway analyses, we identified several vital pathways involved in HCC infected with HCV. The dotplot picture showed that differential mRNAs were significantly enriched in eight pathways (P < 0.05), including the activated pathway-MAPK signalling pathway ( Figure 1B). Then, the Venn diagram showed that there were 27 common mRNAs between differential expression mRNAs in JFH1-infected Huh-7.5.1 cell and MAPK signalling pathway related mRNAs ( Figure 1C). These common genes are showed in Table S2. STRING online retrieval site was used to uncover the association between these genes, and c-Jun turned out to be in the centre of 27 intersecting genes, which indicated that it played vital and stable roles in HCV infection ( Figure 1D). The c-Jun mRNA expression level was higher in hepatitis and HCC infected with HCV compared with those in normal people ( Figure 1E, P < 0.05).

| Determination of the c-Jun expression during HCV infection in HCC cells
As shown in Figure 2A, showed that the HCV RNA expressed stably at least 3 days after transfection with HCV RNA ( Figure 2D). C-Jun expression level was down-regulated by si-c-Jun ( Figure 2E, P < 0.05).

| There was a targeted relationship between miR-148a-3p and c-Jun
Expression levels of miRNAs were considered to be significantly different if their levels meet the standard |log 2 (FC)|>1 and P. adjust < 0.05. Figure 5A showed the top four miRNAs which were up-regulated in infection HCV group and the top 10 down-regulated miR-NAs including miR-148a-3p. According to the Venn diagram in Figure 5B, there was only one of the miRNAs targeted by c-Jun that was differentially expressed in the host of HCV infection. The potential targeted relationship between miR-148a-3p and c-Jun was predicted by TargetScan and verified by Luciferase Reporter Assay ( Figure 5C). The results revealed that the luciferase activity of cells cotransfected with miR-148a-3p mimics and c-Jun 3'UTR-WT was significantly lower than that of cells cotransfected with miR-148a-3p mimics and c-Jun 3'UTR-MUT. Furthermore, qRT-PCR result revealed that miR-148a-3p level was lower in hepatitis and HCC infection with HCV than that in normal people ( Figure 5D, P < 0.05). The Pearson correlation coefficient showed there was a negative correlation between miR-148a-3p and c-Jun expression in HCC infected with HCV ( Figure 5E, P = 0.0016). We constructed the Ad-HCV core adenovirus for infection Huh-7.5.1 cells to overexpress HCV core protein and subsequently detected the expression of HCV core protein, c-Jun and miR-148a-3p. As shown in Figure S1A-S1D, HCV core protein and c-Jun were up-regulated in Ad-HCV core infected cells, whereas miR-148a-3p was down-regulated. The transfection efficiency of miR-148a-3p was shown in Figure 6A (P < 0.05). MiR-148a-3p expression level was notably elevated in mimics group while decreased in inhibitor group when compared to JFH-1 group. C-Jun mRNA and protein expression levels were inhibited by miR-148a-3p mimics but was facilitated by miR-148a-3p inhibitor ( Figure 6B, P < 0.05). As shown in Figure 6C 13,14 In our study, we focused on hsa-miR-148a-3p, which was lowly expressed in HCV infection host and had a targeted relationship with c-Jun. Numerous studies have been performed to uncover miR-148a-3p was involved in several cancers. Lifers revealed miR-148a, down-regulated in human pancreatic ductal adenocarcinomas, regulated cell survival via CDC25B, a conserved dual specificity phosphatase which was significant in appropriate cell cycle. 23 Similar results were also carried out in gastric cancer and F I G U R E 7 MiR-148a-3p affected cell cycle and apoptosis. A and C, MiR-148a-3p inhibitor blocked the cell cycle in G1 phase. B and D, Cell apoptosis was promoted by miR-148a-3p mimics. JFH-1, Huh-7.5.1 cell infected with the JFH-1 strain of HCV. *P < 0.05, compared with JFH-1 HCC. 14 Yuan et al suggested that miR-148a had a central role in hepatitis B virus (HBV) infected HCC via HBx, a trans-activating protein related to the HCC development, 24 and miR-148a was found up-regulated in glioblastoma and chordomas. 14 In our study, we figured c-Jun as the target protein of miR-148a, and it was the first time to combine miR-148a and c-Jun. Moreover, overexpression of miR-148a could restrain the expression of c-Jun.

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Finally, we investigated the regulation of miR-148a on cell processes. Data showed that miR-148a could inhibit proliferation, block G0/G1 phase in cell cycle and enhance cell apoptosis. The regulation was same with some other studies. Results of Bhattacharya et al made it clear that reduction in miR-148a led to the restraint of osteosarcoma cell death. 16 Wang et al revealed that miR-148a could suppress proliferation in bladder cancer. 15 Our study displayed a new kind of mechanism of c-Jun overexpres-