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Excessive miR-30a-5p increases the radiosensitivity of hepatoma cells by inhibiting GRP78

Purpose: To determine the effect of miR-30a-5p on hepatoma cell radiosensitivity and elucidate the underlying mechanism. Methods: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to measure miR-30a-5p expression in HepG2 and THLE-3 cells. After 4-Gy X-ray irradiation or miR-30a5p mimic transfection, the miR-30a-5p level in HepG2 cells was determined using qRT-PCR. Luciferase reporter assay was used to confirm the correlation between miR-30a-5p and glucose-regulated protein 78 (GRP78) levels, while the effects of miR-30a-5p on the viability of HepG2 cells were determined using clone formation and 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assays. Apoptotic cells were evaluated by flow cytometry whereas the protein levels of GRP78, B-cell lymphoma-2 (Bcl-2), BCL2-Associated X Protein (Bax), and cleaved-caspase-9 were quantified by immunoblotting. Results: MicroRNA-30a-5p expression was decreased in HepG2 cells but reduced after 4-Gy x-ray treatment, while miR-30a-5p mimic transfection upregulated miR-30a-5p expression (p < 0.05). Cell viability was inhibited after x-ray irradiation or miR-30a-5p mimic transfection and further inhibited by irradiation + miR-30a-5p (p < 0.05). Irradiation or miR-30a-5p transfection triggered cell apoptosis; however, irradiation + miR-30a-5p induced more apoptosis, upregulated Bax and cleaved-caspase-9 expression, and reduced Bcl-2 expression (p < 0.05). MicroRNA-30a-5p also suppressed GRP78 expression. Conclusion: MicroRNA-30a-5p may enhance HCC x-ray radiosensitivity by inhibiting GRP78., and may be useful in developing treatment strategies for HCC patients


INTRODUCTION
Hepatocellular carcinoma (HCC) is the most common liver cancer and fourth most common cause of cancer-related death worldwide.
Ultrasonic monitoring every six months, with or without alpha fetoprotein, is associated with improved early detection and overall survival rates [1]. Additionally, HCC is relatively resistant to chemotherapy, and surgery remains the only realistic therapeutic option for patients with HCC [2]. However, because of the limitations of detection, HCC is difficult to detect early, leading to fewer than 40% of patients being eligible for surgery [2]. Therefore, an effective detection strategy with higher sensitivity is necessary for early diagnosis, dynamic monitoring, and drug screening for HCC.
MicroRNAs (miRNAs), a class of short RNA molecules, can modulate posttranscriptional gene expression by modulating mRNA degradation or mRNA translation [3]. Compelling evidence has confirmed that miRNAs are essential in radiation therapy, and miR-21 may be a new promising target in cancer radiation therapy [4]. Additionally, miR-302 replacement therapy in breast cancer promotes breast cancer cell sensitivity to ionizing radiation [5]. Furthermore, miRNAs are potential factors that mediate radiosensitivity in HCC. Although miR-30a-5p represses HCC cell growth [6], its role in radiosensitivity of hepatoma cells remains elusive. Therefore, this study investigated the effect of miR-30a-5p on the sensitivity of HCC cells to interventional radiation therapy to develop a novel target for improving the radiotherapy of HCC. Glucose-regulated protein 78 (GRP78) is a key regulator in the tumor microenvironment, and it is associated with cancer cell growth, apoptosis inhibition, immune escape, metastasis, and angiogenesis. Additionally, anti-GRP78 autoantibodies have been used as serological markers for HCC diagnosis [7], and GRP78 is considered as a novel contributor to the acquisition of resistance to sorafenib in HCC [8]. Unfortunately, the relationship between miR-30a-5p and GRP78 remains unclear. Thus, we investigated the effects of miR-30a-5p on the radiosensitivity of HCC and the underlying mechanism.

Real-time fluorescent quantitative PCR
TRIzol ® (Invitrogen, USA) was used for total miRNA extraction from THLE and HepG2 cells, and the miRNA was elongated using E. coli poly A polymerase. After polyadenylation, the miRNA was reverse transcribed into cDNA using an miRNA 1st Strand cDNA Synthesis Kit (by stemloop) (MR101-01; Vazyme Biotech, Nanjing, China), following the manufacturer's recommendations. Next, HiScript II Q Select RT SuperMix for qPCR (R232-01) and AceQ Universal SYBR qPCR Master Mix (Q511-02) were used for PCR using the following parameters: 95 °C for 60 s, with 35 cycles of 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 sec. U6 was the internal reference for miR-30a-5p, and glyceraldehyde-3-phosphate dehydrogenase served as a control for GRP78. The relative levels of GRP78 and miR-30a-5p were quantified using the 2 -ΔΔCT method [9]. The specific primer sequences are shown in Table 1.

Clone formation assay
HepG2 cells (5 × 10 3 cells/well) were seeded in 6-well plates and incubated at 37°C overnight. After 4-Gy X-ray irradiation, the cells were cultured in an incubator at 37°C for 9 days. Subsequently, the cells were fixed with 1 mL of methanol for 10 min, and then the fixative was discarded. Next, 1 mL of Giemsa staining solution (g1015; Solarbio Technology, Beijing, China) was added to cells for 20 min. The colony formation rate = (number of clones/number of cells inoculated) × 100%.

Determination cell viability
After transfection and 4-Gy X-ray irradiation, HepG2 cells at 1 × 10 5 cells/mL were incubated for 24, 48, and 72 h at 37°C. Next, 20 μL of MTT (ab211091; Abcam, USA) was added, the cells were cultured for 4 h at 37°C, and then 100 μL of DMSO was added. The absorbance was measured at 570 nm.

Dual luciferase reporter assays
TargetScan (Release 7.1) and miRDB were used to obtain the binding sites of miR-30a-5p and the GRP78 3ʹ-UTR. HepG2 cells at of 5 × 10 3 cells/well were seeded in 6-well plates overnight.

Annexin V / PI assay
Apoptotic cells were measured using the Annexin V/PI kit (ac12l033; life-ilab Biotechnology, Shanghai, China) according to the manufacturer's instructions. HepG2 cells at 1 × 10 5 cells/well were placed in 6-well plates and then were stained with Annexin V and PI for 15 min in the dark. Apoptotic cells were assessed by FACS Caliber flow cytometry (BD Bioscience).

Statistics
Statistical analysis was performed using Graphpad Prism 8 software (La Jolla, CA, USA). The data of each group were presented as means ± standard deviation. Differences between groups were compared using t test, and one-way analysis of variance was employed to compare among multiple groups. P < 0.05 was considered statistically significant.

DISCUSSION
HCC is a common malignancy, and radiotherapy is an important strategy to treat advanced HCC [10]. The enhancement of radiosensitivity and improvement of radiation efficacy have been research hotspots recently. It is essential to explore the mechanism of radiosensitivity in HCC cells and identify key genes. In the present study, 4-Gy X-ray irradiation or miR-30a-5p transfection was performed in HepG2 cells, followed by cell viability and apoptosis assays. Furthermore, luciferase reporter assays were performed.
MiR-30a-5p is downregulated in various malignancies, is markedly reduced in HCC tissues compared with that in non-cancerous liver tissues, and inhibits liver cancer cell proliferation [11]. The present study demonstrated that miR-30a-5p in HepG2 cells is decreased compared with that in human normal liver cells, indicating that miR-30a-5p is essential in the occurrence and development of HCC.
The abnormal expression of miRNAs is not only implicated in the growth and apoptosis of tumor cells but also has a close relationship with the radiosensitivity of cancers [12]. Liu and colleagues demonstrated that miR-1271-5p reduced cell proliferation but increased radiosensitivity in SMMC-7721 and HuH-7 cells, effects that were reversed by cyclin-dependent kinase 1 [13]. Another study revealed that miR-30a-3p inhibited esophageal carcinoma (EC) cell metastasis and invasion and promoted the radiosensitivity of EC cells [14]. In addition, a previous study showed that hypoxia led to decreased levels of miR-30a and miR-205 in prostate cancer. Furthermore, miR-30a and miR-205 enhanced the sensitivity of prostate cancer cells to irradiation by regulating TP53INP1 [15]. In the current study, 4-Gy X-ray irradiation significantly repressed miR-30a-5p expression in HepG2 cells. X-ray irradiation or miR-30a-5p significantly suppressed cell viability and colony formation, which were further decreased by X-ray irradiation + miR-30a-5p. In addition, X-ray irradiation + miR-30a-5p transfection induced apoptosis of HepG2 cells compared with X-ray irradiation or miR-30a-5p transfection. Thus, miR-30a-5p inhibits cell viability of X-ray-treated hepatoma cells, enhances apoptosis, and increases radiosensitivity.
Expression of GRP78 was positively correlated with cancer progression, tumor size, and poor prognosis. Recently, Chen found that forkhead box P-2 (FOXP2) promoted tumor proliferation and metastasis by modulating GRP78 in triplenegative breast cancer [16]. Furthermore, miR-495-3p exerted inhibitory effects on multidrug resistance by regulating autophagy by suppressing the GRP78/mTOR pathway in gastric cancer [17]. Another study showed that miR-495 increased the sensitivity of nasopharyngeal carcinoma cells during radiotherapy by modulating GRP78 [18]. The present study suggested that GRP78 expression was increased in HepG2 cells. Furthermore, miR-30a-5p targeted the GRP78 3ʹ-UTR region and inhibited GRP78. Additionally, transfection with miR-30a-5p decreased GRP78 expression, suggesting that miR-30a-5p repressed GRP78 and enhanced the radiosensitivity of X-raytreated HCC cells.

CONCLUSION
MiR-30a-5p enhances HCC radiosensitivity to xray by inhibiting GRP78 expression. This finding suggests a new approach to improve radiotherapy in patients with HCC.