miR-643 suppresses cell invasion and radioresistant of lung cancer through RAF1

RAF1 (c-raf) has been known as an important tumor-promoter in many cancers. However, the regulatory mechanisms affecting RAF1 expressions are rarely reported. This study aimed to predict the candidate miRNAs of RAF1 gene and verify their functions in the progression of lung cancer. The expression of miR-643 was detected by reverse transcription polymerase chain reaction (RT-PCR). A dual-luciferase report system was used to verify the relationship between miR-643 and RAF1. RT-PCR and Western blot were used to analyze the regulatory relationship between miR-643 and RAF1. Transwell chamber, scratch, and monoclonal tests showed that miR-643 affected the proliferation, migration, and radiosensitivity of H1299 and A549 cells by targeting the RAF1 gene. The expression of miR-643 in lung cancer cells was lower than that in the bronchial epithelioid cells (CRL-2741), and luciferase reporter experiment confirmed that miR-643 targeted RAF1. MiR-643 overexpression decreased the RAF1 expression, thereby decreasing cell migration, proliferation, and radiation resistance through the AKT/nuclear factor- κ B pathway. miR-643 in lung cancer cells could inhibit cell proliferation, invasion, and metastasis and increase the radiosensitivity by downregulating RAF1.


| INTRODUCTION
RAF1 (c-raf) is an important part of Raf in the RAS/Raf/mitogenrelated signaling pathway, which participates in the regulation of multiple signaling pathways. 1,2 RAF1 phosphorylation and activation of the Ras associated signaling pathway play important roles in cell cycle, proliferation, apoptosis, and migration. MiRNAs are involved in cell differentiation, development, metabolism, proliferation, and withering, promoting or preventing the tumor progressions. [3][4][5][6] Here, the deregulations of miRNAs are closely related to the aggression of non-smallcell lung carcinoma (NSCLC) cells. The high expressions of miR- 21,7 miR-141, 8 miR-221, 9 and miR-222 10 drive the tumorigenesis and increase the treatment resistance of NSCLC cells. The downregulation of miR-128 expression showed negative correlation with the pathological stage of NSCLC, and exaggerated the metastasis of lung cancer cells. 11 A decreased miRNA-29 expression is associated with the poor prognosis of NSCLC. 12 Plasma miRNAs are used to predict and diagnose early non-small cell lung cancer, [13][14][15][16] predict the prognosis of NSCLC patients, 17 and inhibit the growth of lung cancer. 18 miR-34 inhibits the transformation of normal lung cells into cancer cells and increases the radiosensitivity of lung cancer cells. 18 MiR-1246 increases the radiation resistance of lung cancer cells by targeting DR5. 19 miR-185-5p plays a role in the radiosensitization of nasopharyngeal carcinoma by targeting the Wnt2B pathway. 20 But in lung cancer cells, RAF1-related miRNAs are rarely reported.
This study aimed to predict the candidate miRNAs of RAF1 gene, verify the target relationship at the cellular level, and explore the correlation of RAF1 gene and its regulation by miR-643 (acquired online bioinformatics software) in lung cancer cell proliferation, invasion, metastasis, and radiosensitivity.

| qRT PCR assays
Total RNA of cells was extracted using TRIzol (Invitgen, USA) and synthesized first-strand cDNA using the PrimeScript First-Strand cDNA Synthesis kit (Takara, China). The SYBR-Green qRT-PCR was performed on an ABI7500 system (Applied Biosystems, USA). Three copies of each experiment.

| Wound healing assay
The method has described in our previous work. 21

| Monoclonal formation experiment
The NC-mimic and miR-643-mimic groups received 0, 2, 4, and 6 Gy X-ray irradiation, and the cell clone formation was observed after

| Western blot assays
The proteins were extracted and measured using RIPA (KeyGEN, China) and BCA protein assay kit (KeyGEN, Nanjing, China). Twenty micrograms of proteins was electrophoresed and subsequently transferred to a PVDF membrane. The membrane was blocked with 5% After washing three times, the membrane was incubated with secondary antibodies for 1 h and visualized by enhanced chemiluminescence detection reagent.

| Statistical analysis
The results were analyzed by SPSS20.0 software and expressed as the mean ± standard deviation. The comparisons of two groups were analyzed using Student's t-test, The statistical difference among multiple groups was evaluated by one-way ANOVA. It is considered statistically significant at the moment that P < 0.05.

| miR-643 expression was downregulated in A549 and H1299 lung cancer cells
We utilized Starbase (https://starbase.sysu.edu.cn/), miRada (http:// www.microrna.org/), and Targetscan (http://www.targetscan.org/) to determine the potential miRNAs that could target RAF1. Of the results provided by the Bioinformatics website, such as miR-654-3p, miR-485-5p, and miR-643, we focused on miR-643 because of its potential as a tumor suppressor in various cancers. The results illustrated that RAF1 expression decreased sharply at the mRNA and protein levels in the miR-643 mimc groups ( Figure 1A,B). Furthermore, the dual-luciferase assays showed that miR-643 + RAF1-3 0 UTR could significantly reduce luciferase activity ( Figure 1C), whereas no significant decrease was observed in the luciferase activity of the mutated RAF1-3 0 UTR group ( Figure 1D). Next, we detected the expressions of miR-643 in the cells. The results exhibited low expression of miR-643 in H1299 cells with a strong metastatic ability and high expressions in A549 and H827 cells with a weak metastatic ability ( Figure 1E). These data indicated that miR-643 was downregulated in lung cancer cells and directly targeted RAF1.

| miR-643 decreases pulmonary carcinoma cell invasion and migration and promotes its radiosensitivity
The gain-of-function analysis has been used to investigate the role of miR-643 performed in progression of the lung cancer cells. We significantly decreased compared with the controls (Figure 2A,B).
We also used clone survival assay to explore the effects of miR-643 on cell sensitivity to irradiation. After increasing miR-643 expressions, A549 and H1299 cells showed lower the survival scores compared with controls ( Figure 3A; Table 1). Additionally, the data of flow cytometry indicated that the miR-643 up-regulation enhanced the proportion of lung cancer cell apoptosis after irradiation ( Figure 3B,C).

| DISCUSSION
In previous studies, we observed that RAF1 was differentially expressed in tumor tissues of patients with different NSCLC and associated with the clinical resistance of radiotherapy in patients with NSCLC. 21 In this study, we used various biological software online and dual-luciferase assays to identify the interaction between miR- caspase-3. These data indicated that miR-643 induced cell apoptosis after irradiation via the mitochondrial signaling pathway.

| CONCLUSION
In summary, target relationship was found between miR-643 and were generated in-house and that no paper mill was used.

ACKNOWLEDGMENTS
This program was funded by Jiangsu "333" project (20180278) and Research Development Fund of Kangda College, Nanjing Medical University (KD2018KYJJYB033).