1. MiR125a-5p was reduced in sorafenib-sensitive HCC
Previously, we selected 16 patients with adjuvant sorafenib treatment after liver resection for hepatocellular carcinoma. Among them, 10 patients (No.1–10) were found to have early tumor recurrence within 1 year after surgery, and 6 patients (No.11–16) were without recurrence. Differentially expressed microRNAs were explored by applying microRNA array in paraffin-fixed tumor tissue, and the top 16 differentially expressed microRNAs were found to be statistically significant. Among these 16 microRNAs, miR125a-5p was the most significant one (Fig. 1. A-B). Further comparison of sorafenib-sensitive and sorafenib-resistant tumor by real-time PCR analysis confirmed that sorafenib-sensitive tumors possessed lower expression of miR125a-5p (Fig. 1.C).
We compared patients’ survival by classification of high and low expression of microRNA by in situ hybridization (ISH) of the tissue microarray. The ISH staining intensity was classified by the median value and it was found that higher expression of miR125a-5p was associated with better OS (p = 0.026) and RFS (p = 0.020) (Fig. 1.D-F). For patients with higher or lower expression of miR125a-5p, the median overall survival were 50.1 months and 30.2 months, respectively (Fig. 1.D). And the median recurrence-free survival in higher/lower miR125a-5p were 23.4 months and 12.3 months, respectively (Fig. 1.E). The staining tensity was showed in Fig. 1.F.
Comparison of clinicopathological factors between high and low expression of miR125a-5p revealed that patient whose tumor has higher miR125a-5p expression was associated with less severe liver inflammation and better tumor biology, as indicated by relatively lower ALT(p = 0.029), lower AST (p = 0.027) and lower AFP (p = 0.013) and lower CA19-9 (p = 0.029) (Table 1). However, the expression of miR125a-5p was not related to patient age, sex and tumor factors including macrovascular invasion, microvascular invasion and tumor staging.
Table 1
Comparison of clinicopathological factors between HCC patients with miR125a-5p (+) and miR125a-5p (-).
Variables |
miR125a-5p (-) (n = 75) |
miR125a-5p (+) (n = 68) |
P-value |
Gender (Male/Female) |
62/13 |
53/15 |
0.477 |
Age (year) (Mean ± SD) |
56.1 ± 12.2 |
55.1 ± 11.9 |
0.634 |
Tumor size (cm) (Mean ± SD) |
6.0 ± 4.1 |
6.1 ± 4.5 |
0.404 |
BCLC (0/A/B/C) |
8/55/5/7 |
2/55/5/6 |
0.342 |
MaVI (Yes vs. no) |
68/7 |
61/7 |
0.847 |
MiVI (Yes vs. no) |
34/41 |
33/35 |
0.702 |
AFP (≤ 20 vs. >20 ng/mL) |
26/48 |
37/29 |
0.013 |
CA199 (≤ 37 vs. >37 U/mL) |
59/15 |
62/5 |
0.029 |
HBeAg (Yes vs. no) |
12/63 |
9/59 |
0.641 |
Cirrhosis (Yes vs. no) |
47/28 |
38/30 |
0.409 |
ALT (≤ 40 vs. >40 U/L) |
42/33 |
49/18 |
0.034 |
AST (≤ 40 vs. >40 U/L) |
44/31 |
51/16 |
0.027 |
Note: MaVI, Macroscopic Vascular Invasion. MiVI, Microscopic Vascular Invasion. AFP, α-fetoprotein. CA19-9, Carbohydrate Antigen 19 − 9. HBeAg, Hepatitis B virus e Antigen. ALT, Alanine Aminotransferase. AST, Aspartate Aminotransferase. |
2. MiR125a-5p over expression led to sorafenib resistance in vitro
We examined miR125a-5p level in 7 different cell lines and found that SMMC7721 expressed the highest level of miR125a-5p and the LM3 expressed the lowest level of miR125a-5p (Fig. 2A). Previously we have testified that the IC50 for sorafenib on SMMC7721 was significantly higher than that on LM3, indicating that SMMC7721 was more resistant to sorafenib compared to LM3. Then we constructed cell lines with miR125a-5p overexpression in LM3 (LM3-OE) and miR125a-5p knockdown in SMMC7721 (SMMC7721-KD). And we examined the expression level of miR125a-5p in SMMC7721-NC, SMMC7721-KD, LM3-NC and LM3-OE before and after sorafenib treatment and we found that sorafenib upregulated miR125a-5p in SMMC7721-KD cell line and downregulate miR125a-5p in LM3-OE cell line (Fig. 2. B-C).
Proliferation assay showed that LM3-OE become resistant to sorafenib compared with LM3-NC (Fig. 2. D-E), while SMMC7721-KD become more sensitive to sorafenib treatment than SMMC7721-NC (Fig. 2. F-G).
3. MiR125a-5p over expression led to sorafenib resistance in vivo.
To explore the impact of miR125a-5p on tumor growth in vivo, we constructed xenograft tumor model by injecting two different kinds of tumor cells in the right flank of BALBc nude mice. Tumor growth in mice was monitored twice weekly.
In SMMC7721-NC model, sorafenib did not significantly inhibit tumor growth until the last week (p = 0.0385, Fig. 3A-B); however, in SMMC7721-KD model, the tumor grew faster than tumor of SMMC7721 model and was significantly inhibited by sorafenib (p = 0.0051, Fig. 3A-B).
In LM3-NC model, sorafenib significantly inhibit tumor growth (p = 0.0357); however, in LM3-OE model, sorafenib did not significantly inhibit tumor growth through the whole treatment period (p = 0.5793, Fig. 3. C-D). Figure 3E showed the real time fluorescence imaging of BALBc mice of LM3 model. So, tumor with higher expression of miR125a-5p was more resistant to sorafenib treatment, which was in accordance with clinical findings.
4. RNAseq results revealed that STAT3 pathway was downregulated by miR125a-5p
To explore the underlying molecular mechanisms, high throughput Affymatrix Array was applied to screening differently expressed genes and pathways in tumor tissues applied in the in vivo study. Ingenuity Pathway Analysis (IPA) analysis showed that acute phase response signaling and STAT3 pathway were found to be significantly suppressed by miR125a-5p in tumor samples of LM3-OE compared with LM3-WT (Fig. 4B). Furthermore, IL-6R was significantly down-regulated by miR125a-5p in LM3-OE compared to LM3-WT. IL-1B and related genes were revealed to be most significantly inhibited by miR125a-5p, together with 26 simultaneously inhibited genes, such as REL, FOS, IL1A, NRP1 etc. (Fig. 4C).
The differentially expressed genes and diseases and functional relationship revealed that the most significantly activated functions including organismal death (Z-score = 2.876), cell death of tumor cells (Z-score = 2.095), while angiogenesis (Z-score=-3.521), cell viability of tumor cell lines (Z-score=-3.513) were significantly inhibited by miR125a-5p over expression. And the regulation network ranking first in this regulation effect analysis shows that the data set may be due to the cell move of the regulators IL1R1, MAP3K8, miR-155-5p, miR-30c-5p, PTPRJ, TAC1, TBK1, ZFP36 through APOE, CTSS, CUX1, CYP51A1, DSG2, FOS, IL1A, IL6, MET, PTGS2, RHEB, SAA1, SEL, SLC7A11, TFRC and other genes of myeloid cells, cell viability, cellular infiltrations by leukocytes, growth of connected tissue, secondary tumor, vasculogenesis have inhibitory effects (Fig. 4D). These findings confirmed that miR125a-5p is a tumor suppressor. The expression trend of each molecule in the classical pathway were shown in Fig. 4E-F.
5. STAT3 and HTATIP2 are direct targets of miR125a-5p
Website for microRNA target prediction (http://www.microrna.org) showed that miR125a-5p directly inhibit STAT3 (Fig. 5.A) and HTATIP2 (Fig. 5.B). The inhibition effect of miR125a-5p was further confirmed by TargetScan Human 8.0 (https://www.targetscan.org/vert_80) (Fig. 5.C-D). The luciferase reporter experiments were also designed. The 3’-UTR segments of STAT3 predicted to interact with miR125a-5p were amplified by PCR and inserted into pGL3 vector immediately downstream from the stop codon of luciferase, and the intensity of luciferase of STAT3 promoter was significantly reduced by miR125a-5p (Fig. 5E).
Furthermore, the upregulation of IL-6R, STAT3 and HATITP2 after knocking down of miR125a-5p was confirmed by PCR in SMMC7721 cells (Fig. 5F), and the downregulation of IL-6R, STAT3 and HATITP2 were confirmed after over expression of miR125a-5p in LM3 cells (Fig. 5.G). Western blotting was also applied to detect the inhibitory effect of miR125a-5p to STAT3 and HTATIP2. AG490 was a small molecule specifically inhibit STAT3. After miR125a-5p knocking down in SMMC7721-KD cell lines, STAT3, pSTAT3 and HTATIP2 were significantly upregulated, while sorafenib and AG490 were unable to downregulate STAT3, pSTAT3 and HTATIP2 (Fig. 5H). And after miR125a-5 overexpression in LM3-OE cell lines, sorafenib and AG490 inhibited STAT3, pSTAT3 and HTATIP2 to a more significant extent (Fig. 5I).