MiR-381 enhances the sensitivity of non-small cell lung cancer to radiotherapy by targeting ROCK2 to regulate NF-κB signaling pathway

We investigated the effect of miR-381 on the sensitivity of non-small cell lung cancer (NSCLC) to radiotherapy, and examined its possible mechanism. NSCLC A549 cells and miR-381 overexpression and gene silencing cell lines were treated with radiotherapy. The cell proliferation was tested by CCK-8 assay and colony formation. Flow cytometry and TUNEL were used to detect the cell apoptosis. The expression of nuclear factor kappa B (NF-κB) signaling pathway related proteins were detected by western blot. NF-κB signaling pathway activator and inhibitor cell lines were further constructed and the above experiments were repeated. Double luciferase assay was used to verify the target of miR-381. Furthermore, a nude mouse xenograft model was constructed and treated with radiotherapy. The tumor volume and tumor weight were measured. The expression of PCNA protein in tumor tissues was observed by immunohistochemistry. The apoptosis related proteins in tumor tissues were detected by western blot. cell and contrary in that signaling pathway activator A number of studies found that NF-κB signaling pathway plays highly important roles in cell proliferation, apoptosis, and drug resistance. A variety of coalesce on NF-κB activation, can in turn mediate varied transcriptional programs. this study, the protein expression of P65, IκBα, and their phosphorylated were detected by western blot. The results found that after silencing miR-381, the expression of p-P65/P65 was increased and the expression of p-IκBα/IκBα was reduced, which indicated that miR-381 overexpression could inhibit NF-κB signaling pathway. To further dene whether miR-381 overexpression enhances the sensitivity of NSCLC to radiotherapy through the NF-κB signaling pathway, the pathway activator and inhibitor were used. The results showed that the addition of NF-κB inhibitor evidently decreased the proliferation ability and increased cell apoptosis. All those results veried that miR-381 overexpression enhances the sensitivity of NSCLC to radiotherapy by inhibiting NF-κB signaling pathway.


Introduction
At present, there are 1.2 million new cases of lung cancer every year and one person dies from lung cancer every 30 seconds worldwide. 1,2 Lung cancer is clinically divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) by histological methods. Non-small cell lung cancer accounts for about 85% of all lung cancer. 3 Additionally, there are differences between males and females in the incidence of lung cancer, smoking prevalence and life expectancy. 4,5 Only a small percentage of NSCLC patients are diagnosed at an early stage, when the tumor can be treated by surgical resection. 2 About 75% of NSCLC patients present with a locally advanced or metastatic disease at the time of diagnosis. 2 Despite treatment advances, conventional chemotherapy and radiation therapy are still the main treatments for patients with lung cancer.
MicroRNAs (miRNAs) is a type of non-coding RNA with a size of about 22 nt, which has a wide range of biological functions such as proliferation, invasion, metastasis and cell survival. [6][7][8] MiRNAs can speci cally bind to mRNA to recruit related RNases and cause mRNA degradation, thereby blocking the expression of protein-encoding genes and affecting its biological function. 9 Numerous studies have demonstrated that miRNAs involve in the occurrence, development, invasion, and metastasis of various human malignant tumors. 10,11 Moreover, growing amount of evidences have suggested that miRNAs have signi cant differential expression in lung cancer patients with different prognosis and benign lung lesions. 12 One of those miRNAs, miR-381 was shown to serve as a tumor suppressor in some cancers. 13,14 Rho-associated protein kinase 2 (ROCK2), a member of the serine/threonine AGC kinase family, is a regulator that can regulate cell proliferation, invasion and metastasis. 15 Study has reported that ROCK2 may be could affect cancer treatment outcomes. 16 Furthermore, Yilin Xie et al. found that miR-381 could serve as a tumor suppressor by targeting ROCK2. 17 Radiotherapy is the main treatment for most cancers. Improving the sensitivity of cancer cells to radiotherapy has an important role for the treatment of various malignant cancers. Therefore, it is signi cant to study the effects and potential underlying mechanisms of miR-381 on the sensitivity of NSCLC to radiotherapy.
In this study, we hypothesize that miR-381 overexpression could enhance the sensitivity of NSCLC to radiotherapy, and further sought to verify whether miR-381 improves the sensitivity of NSCLC to radiotherapy through regulating nuclear factor kappa B (NF-κB) signaling pathway.

Cell transfection and grouping
Cells were passaged and inoculated on a 6-well plate. When the cells reached 70% con uence, transfection was performed according to the lentiviral transfection instruction (Shanghai Jikai Biotechnology Co., Ltd., Shanghai, China). The cells were divided into 6 groups: i) blank control group (BC), no treatment; ii) radiotherapy group (Rad), cells were exposed to a single dose of X-rays using a linear accelerator (RadSource, Suwanee, GA, USA) at a dose rate of 1.15 Gy/min and 160 kv X-ray energy, 18 no transfection; iii) miR-381 overexpression negative control group (NC-1), cells were radiotherapy treatment and transfected with miR-381 scramble; iv) miR-381 overexpression group (miR-381), cells were radiotherapy treatment and transfected with miR-381 mimics; v) miR-381 silencing negative control group (NC-2), cells were radiotherapy treatment and transfected with miR-381 inhibitor negative control; and vi) miR-381 silencing group (si-miR), cells were radiotherapy treatment and transfected with miR-381 inhibitor.
Cell counting kit (CCK)-8 assay The proliferation ability of cells was performed by the CCK-8 assay kit (Dojindo, Japan) according to the manufacturer's protocol. Logarithmic growth phase cells were cultured in 96-well plates at a density of 2×10 4 cells/ml, 100 μl per well. After culture for 24 h, 48 h, 72 h, and 96 h, 10μl of CCK-8 solution was added into plates and then followed with incubation for 4 h at 37˚C, 5% CO 2 . The optical density (OD) was measured at 450 nm.

Colony formation assay
Logarithmic growth phase cells were digested with 0.25% trypsin and adjusted to 250 cells/ml. 2 ml/well cells were cultured in a 6-well plate at 37°C, 5% CO 2 for 2-3 weeks and the fresh medium was changed every 3 days. The cells were xed in methanol and each well was added with 1mL Ji Giemsa working uid and stained for 30 min. After washed twice with ultrapure water, the record was imaged by a camera.
Flow cytometry 24 h after transfection, the cells were collected and resuspended with pre-chilled 1× PBS, and centrifuged at 1000 rpm for 5-10 min. After washing, 300 μl 1× binding buffer was added to the cells for suspension, and 5 μl Annexin V-FITC was added. The cells were mixed and incubated in dark for 15min. Then, 5 μl propidium iodide (PI) was added and incubated in the dark for 5 min. 200 μl 1× binding buffer was added prior to analysis using ow cytometry (Beckman Coulter, Brea, CA, USA). CellQuest software (BD Biosicences, San Diego, USA) was used to analyze the results.   Construction and grouping of lung cancer xenograft model The lung cancer cells A549 in logarithmic growth phase were digested with 0.25% trypsin, collected and counted. The cell concentration was adjusted to 2.5×10 6 cells/ ml, and 0.2 ml was inoculated into the soft skin of the right forelimb back of the nude mouse. 36 mice were randomly divided into 6 group (n=6): i) blank control group (BC); ii) radiotherapy group (Rad); iii) miR-381 mimic group (miR-381); iv) Radiotherapy + miR-381 overexpression negative control group (NC-1); v) Radiotherapy + miR-381 silent group (si-miR), and vi) Radiotherapy + miR-381 silent negative control group (NC-2). One week later, except for the control group, the other nude mice were irradiated with 6MV X-rays at a dose rate of 2 Gy/min. A lead plate with a distance of 100 cm and the latter 3 mm covered other parts of the nude mice, and the dose was 10 Gy.

Tumor volume calculation
The tumor's long diameter (L) and short diameter (W) were measured every week with vernier caliper. The tumor volume (V) = (L x W 2 )/2. Tumor was measured on a weekly basis for 4 weeks and the tumor growth curve was drawn. 4 weeks later, nude mice were anesthetized by intraperitoneal injection of 0.6% sodium pentobarbital (40 mg/kg, New Asiatic Pharmaceutical, China) and sacri ced by cervical dislocation.

Immunohistochemistry
After conventional sectioning of the tumor tissue, the slices were baked, dewaxed with xylene, and sequentially hydrated with a gradient ethanol solution. 3% H 2 O 2 methanol solution was used to inactivate processing for 20 min. The slices were heated with citrate buffer (pH 6.0) for 10 min and sealed with 5% BSA for 20 min. After blocking with 5% goat serum (Gibco, USA) for 20 min, the slices were incubated with rabbit anti-human PCNA (1: 500, orb251877, Biorbyt, Cambridge, UK) polyclonal antibody overnight at 4 °C. Then, the goat anti-rabbit IgG (1: 1000, ABIN101988, antibodies-online, Germany) labeled with horseradish peroxidase was used for secondary antibody incubation. 3,3'-Diaminobenzidine staining, hematoxylin counterstaining, dehydration and sealed. The expression of PCNA in each group was observed under a × 400 light microscope (Olympus, Japan) and count using AperioImagescope 11.1 software.

Double luciferase reporter assay
The wild type and mutant 3'UTRs of ROCK2 were ampli ed in pGL3/luciferase vector (Promega, Madison, WI, USA) and cloned to the downstream of the luciferase gene. The cells were tested for luciferase activity using a dual luciferase reporter system (Promega) 48 h after transfection according to the instructions.
Statistical methods SPSS19.0 statistical software was used to analyze the data. The results were expressed as mean ± standard deviation (SD). Data analysis between the two groups using t test. Means of analysis between multiple groups were analyzed by single factor analysis of variance (ANOVA), and subsequent analysis was performed by LSD test. P < 0.05 indicates that the difference is statistically signi cant.

Results
Effects of radiotherapy treatment on the proliferation and apoptosis of A549 cells As shown in Figure 1, compared with BC group, radiotherapy treatment signi cantly inhibited the proliferation and promoted apoptosis of A549 cells (p < 0.05). Meanwhile, the mRNA expression of miR-381 in Rad group was remarkably higher than the BC group (p < 0.05).

Effects of miR-381 on the proliferation of A549 cells
Contrasted with Rad group, the level of miR-381 mRNA in miR-381 group was remarkably raised, and that was remarkably declined in si-miR group (p < 0.05, Figure 2A). Contrasted with Rad group, the cell proliferation ability of miR-381 group was remarkably reduced, and the cell proliferation ability of si-miR group was signi cantly increased (p < 0.05, Figure 2B and 2C). Moreover, the cell proliferation ability of si-miR group was signi cantly higher than the miR-381 group (p < 0.05). The results revealed that miR-381 overexpression could enhance the sensitivity of NSCLC to radiotherapy.

Effects of miR-381 on the apoptosis of A549 cells and NF-κB signaling pathway
As shown in Figure 3, contrasted with Rad group, the apoptotic rate and apoptosis index of miR-381 group were remarkably raised, and the expression of p-P65/P65 was remarkably declined, moreover, the expression of p-IκBα/IκBα was remarkably raised (p < 0.05). However, the results of the si-miR group were exactly opposite. Contrasted with miR-381 group, the apoptosis rate and apoptosis index of si-miR group were signi cantly reduced, and the expression of p-P65/P65 was signi cantly increased, moreover, the expression of p-IκBα/IκBα was signi cantly reduced (p < 0.05). Those results suggested that miR-381 overexpression could enhance the sensitivity of NSCLC to radiotherapy and inhibit NF-κB signaling pathway.

miR-381 regulates the proliferation of A549 cells by regulating NF-κB signaling pathway
To verify the relationship between miR-381 and NF-κB signaling pathway, NF-κB signaling pathway activator and inhibitor cell lines were further constructed. Figure 4 shown that compared with BC group, the cell proliferation ability of other groups was signi cantly reduced (p < 0.05). Simultaneously, compared with Rad group, the cell proliferation ability of miR-381 and PDTC groups was dramatically decreased (p < 0.05). However, compared with miR-381 and PDTC groups, the cell proliferation ability of miR + PMA group was signi cantly increased (p < 0.05). Those results indicated that miR-381 overexpression enhances the sensitivity of NSCLC to radiotherapy by inhibiting NF-κB signaling pathway.

miR-381 regulates the apoptosis of A549 cells by regulating NF-κB signaling pathway
Furthermore, as exhibited in Figures 5, compared with BC group, the cell apoptosis rate and apoptosis index of other groups were signi cantly increased, and the expression of p-P65/P65 was signi cantly reduced, moreover, the expression of p-IκBα/IκBα was signi cantly increased (p < 0.05). Compared with Rad group, the apoptotic rate and apoptosis index of miR-381 and PDTC groups were signi cantly increased, the expression of p-P65/P65 in the NF-κB signaling pathway was dramatically decreased, and the expression of p-IκBα/IκBα was signi cantly increased (p < 0.05). However, the results of the miR + PMA group were exactly opposite. All those results veri ed that miR-381 overexpression enhances the sensitivity of NSCLC to radiotherapy by inhibiting NF-κB signaling pathway.

Effects of miR-381 on NSCLC xenografts
A nude mouse xenograft model was further constructed and treated with radiotherapy ( Figure 6).
Compared with BC group, the tumor growth rate in the other groups was signi cantly slowed, tumor volume, weight, and the percentage of PCNA-positive cells were signi cantly decreased, while, the expression of apoptosis-related proteins were signi cantly increased (p < 0.05). Compared with Rad group, the miR-381 group has a better tumor suppressive effect, but the si-miR group had the opposite effect (p < 0.05). Compared with miR-381 group, the tumor growth rate in the si-miR group was signi cantly accelerated, the tumor volume, weight, and the percentage of PCNA-positive cells were signi cantly increased, and the expressions of Bax and Caspase-9 were signi cantly decreased (p < 0.05). All these ndings demonstrated that miR-381 overexpression could inhibit cell tumorigenicity and proliferation, and promote the apoptosis.
ROCK2 is the target of miR-381 As shown in Figure 7, a bioinformatics search identi ed ROCK2 is the target of miR-381. A dual luciferase reporting system was used to further verify whether miR-381 targets ROCK2. The results showed that miR-381 reduced the luciferase activity of ROCK containing WT 3'UTR, but did not decrease the luciferase activity of ROCK containing Mut 3'UTR. Compared with the BC group, the ROCK protein expression was signi cantly increased in radiotherapy group (p < 0.05). Moreover, miR-381 overexpression could reduce the expression of ROCK2 and miR-381 silencing could increase the expression of ROCK2.

Discussions
As a member of miRNA family, miR-381 affects metastasis in some type of cancers. [14][15][16][17] In this study, the potential role of miR-381 in lung cancer was rstly evaluated. We found that the mRNA expression of miR-381 in radiotherapy cells was remarkably higher than that in blank control cells, and the miR-381 overexpression treatment could inhibit proliferation and promote apoptosis of NSCLC. Those results indicated that miR-381 plays a positive role in the treatment of NSCLC. Furthermore, through predicted by databases and dual luciferase assay, we found that ROCK2 is the target of miR-381. The protein expression of the ROCK2 were signi cantly down-regulated after miR-381 mimic transfection and miR-381 silencing could increase the expression of ROCK2.
Cell proliferation is the foundation of organism growth, development, reproduction, and heredity. The cancer progression usually associated with abnormal cells proliferation, migration and invasion.
Uncontrolled proliferation provides a survival advantage of cancer cells to resist conventional chemotherapeutic agents. [19][20][21] In this study, CCK-8 and cloning formation assays con rmed the miR-381 overexpression could inhibit cell tumorigenicity and proliferation, which suggested that miR-381 overexpression could enhance the sensitivity of NSCLC to radiotherapy. Apoptosis is a basic biological phenomenon of cells and plays a necessary role in the removal of unwanted or abnormal cells by multicellular organisms. Apoptosis is the main cause of tumor cell death and is the core mechanism for preventing tumor growth. 22 Flow cytometry and TUNEL staining showed miR-381 overexpression increased the apoptotic level of NSCLC cells. The expression of Bax and Caspase-9 were further evaluated, and the apoptosis related proteins in tumor tissues were increased after miR-381 mimic interference.
The mechanisms involved in the NSCLC have not yet been completely elucidated. A number of studies found that NF-κB signaling pathway plays highly important roles in cell proliferation, apoptosis, angiogenesis, in ammation, metastasis, and drug resistance. [23][24][25] A variety of stimuli coalesce on NF-κB activation, which can in turn mediate varied transcriptional programs. 26 In this study, the protein expression of P65, IκBα, and their phosphorylated were detected by western blot. The results found that after silencing miR-381, the expression of p-P65/P65 was increased and the expression of p-IκBα/IκBα was reduced, which indicated that miR-381 overexpression could inhibit NF-κB signaling pathway. To further de ne whether miR-381 overexpression enhances the sensitivity of NSCLC to radiotherapy through the NF-κB signaling pathway, the pathway activator and inhibitor were used. The results showed that the addition of NF-κB inhibitor evidently decreased the proliferation ability and increased cell apoptosis. All those results veri ed that miR-381 overexpression enhances the sensitivity of NSCLC to radiotherapy by inhibiting NF-κB signaling pathway.

Conclusion
In sum, miR-381 overexpression could inhibit cell tumorigenicity and proliferation, and promote the apoptosis, which suggested that miR-381 overexpression could enhance the sensitivity of NSCLC to radiotherapy. The possible mechanism is related to targeting ROCK2 to suppress NF-κB signaling pathway. Therefore, miR-381 maybe a potential therapeutic agent for further treatment of lung cancer.

Declarations
All manuscripts must contain the following sections under the heading 'Declarations': Ethics approval and consent to participateAnimal experiments were followed the NIH guidelines (NIH Pub. No. 85-23, revised 1996) and have been approved by the Animal Protection and Use Committee of Tianjin Medical University Cancer Institute and Hospital.

Consent for publication
Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests       (D) Immunohistochemistry was used to detect the expression of PCNA; (E) Western blot was used to detect the expression of apoptosis related proteins. *p < 0.05 compared with BC group; #p < 0.05 compared with Rad group; ^p < 0.05 compared with miR-381 group.