The role of miRNA-377 as a tumor suppressor in lung cancer by negative regulation of genes belonging to ErbB signaling pathway

The ErbB signaling pathway plays important role in the pathogenesis of lung cancer. We explored the role of miRNA-377 as a tumor suppressor in NSCLC through silencing of some genes in the ErbB pathway. The targeting effect of miRNA-377 on EGFR, MAPK1, ABL2, and PAK2 was evaluated. The expression levels of these genes and miRNA-377 were surveyed in NSCLC and normal human tissues, Calu-6, and A549 cells. Real-time PCR was used to figure out whether miRNA-377 could decrease the target genes mRNAs in transfected lung cancer cell lines. The effects of miRNA-377 on apoptosis cell and proliferation were analyzed. We showed that miRNA-377 targets EGFR, MAPK1, and PAK2 mRNAs in in-silico and luciferase reporter assay. The expression of miRNA-377 was significantly downregulated in human NSCLC tissues, Calu-6 and A549 cells compared to their controls. We observed a negative correlation between EGFR, MAPK1, PAK2, and miRNA-377 expression in human NSCLC tissues. A significant reduction in EGFR, MAPK1, and PAK2 mRNA levels was detected, following miRNA-377 transfection in Calu-6 and A549 cells. The higher levels of miRNA-377 in Calu-6, and A549 cells induced apoptosis and reduced proliferation, significantly. All these data reveal that miRNA-377 functions as a tumor suppressor in NSCLC and may serve as a potential therapeutic target for the treatment of NSCLC.


Introduction
Lung cancer is the first leading cause of cancer death (18.4% of the total cancer deaths) [1], which is classified into two major groups: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC accounts for approximately 85% of all lung cancers and the treatment efficacy of currently anticancer agents is usually undesirable and patients with advanced diseases scarcely have long life [2][3][4]. Therefore, it is essential to develop an efficient strategy for the treatment of NSCLC. Oncogenic mutations and epigenetic changes disrupt the signaling network like Saba Hashemi, Naghmeh Yari have contributed equally to this work. epidermal growth factor receptor, transforming growth factor-beta, Hippo, phosphoinositide 3-kinase/AKT/mammalian target of rapamycin, Wnt, mitogen-activated protein kinase, nuclear factor kappa-light-chain-enhancer of activated B cells, Janus kinase/signal transducers and activators of transcription, etc. These signals manage cell fate, keeping tumor cells with numerous traits enduring their malicious performance and have the potential to be targeted for anticancer therapy. The ability of normal cells to live and grow depends on participation of growth factor receptors provided with tyrosine kinase activity. However, oncogenic mutations and aberrant epigenetic patterns triggering abnormal activation of receptor tyrosine kinases or their downstream signalling elements are common in cancer [5,6].
The ErbB signaling pathway and its receptors with tyrosine kinase activity performs main roles in the molecular pathogenesis of many types of human cancer [7,8], including lung cancer [9]. The ErbB receptors family has four members, including EGFR (ErbB1), HER2/neu (ErbB2), HER3 (ErbB3), and HER4 (ErbB4). Homo-or heterodimerization of these receptors occurs after ligand binding, leads to activation of downstream signaling cascades like PLCγ, PI3K/AKT, ERK and more with the cell. EGFR mutations are found in some types of lung cancers and this mutation is associated with either HER2 or HER3 activation and triggers downstream signaling pathways like anti-apoptotic signaling [10,11]. Because overexpression of the EGFR occurs in 40-80% of patients with NSCLC, it has been an attractive target for the development of therapeutic agents [12,13]. miRNAs (microRNAs) are short single-stranded non-coding RNAs, containing approximately 18-24 nucleotides that regulate post-transcriptional gene expression by targeting 3′ untranslated regions (3ʹ-UTRs) of mRNAs with their seed sequences (2-7 nucleotides in the 5′ end) [14]. miRNAs, target the mRNAs of many different genes, consequently, these ncRNAs unsurprisingly connect related genes into controlling networks. The networks in which miRNA act their functions perform to supply checks and stabilities on vital regulatory activities. Variations in the several layers of control of genes that permit the cell to repair DNA damage, perform cell division, react to mitogenic signals, shape chromatin, and interact with other cell types could have harmful outcomes. Alterations in these extremely regulated pathways may result in prompt the cell towards unrestricted cell growth and tumorigenicity. The results of many studies shows that miRs are deregulated and involved in different cancer progressions, such as initiation, metastasis, cancer stem cell (CSC) and drug resistance, emphasizing the role of miRs as potential therapeutic targets in cancer [15,16].
Deregulation of several miRNAs, which are involved in various biological and pathological processes, has been found in cancers [17][18][19], making them attractive targets for therapeutic intervention. miRNAs such as miRNA-125a, miRNA-125b, miRNA-199a, miRNA-21, miRNA-1 miRNA-125a-5p and miRNA-145 that mediated regulation of the ErbB signaling [20][21][22] are of special attention due to the important contribution of this pathway to cancer development. These examples of miRNA ErbB signaling network illustrate the role of miRNA regulation and their potential for therapeutic purposes. In the present study, we targeted EGFR, ABL2, MAPK1 and PAK2 to silencing ErbB pathway in NSCLC. We intended to use just one miRNA to silence the ErbB pathway genes and according to the bioinformatics analysis results, miRNA-377 with a high score targeted all four main genes in this pathway.
The aim of this study was to further prospect the participation of miRNA-377 in NSCLC progression, through the analysis of the connection between miRNA-377 expression and four ErbB pathway elements which their deregulation have key role in lung cancer development. In this study, we firstly verified the targeting of miRNA-377 on EGFR, MAPK1, and PAK2, by using bioinformatics analyses. Then we confirmed that miRNA-377 directly targeted EGFR, MAPK1, and PAK2 but not ABL2 by luciferase reporter assay. In next step our results showed that miRNA-377 expression significantly decreased in NSCLC tissue and lung cancer cell lines compared with normal tissue. Furthermore, we found that the mRNAs of EGFR, MAPK1 and PAK2 significantly increased in NSCLC tissue. We showed a significant reduction in EGFR, MAPK1 and PAK2 mRNA levels following miRNA-377 transfection in Calu-6 and A549 cells. Moreover, transfection of miRNA-377 set in motion cell proliferation and apoptosis of lung cancer cells. In conclusion, our findings suggest that miRNA-377, assisting as a tumor suppressor, played an inhibit role in the development of NSCLC by negative regulation of EGFR, MAPK1 and PAK2 expression, thereby play a part in the progress of NSCLC.

miRNA selection
Bioinformatics studies were conducted to select the appropriate miRNA that could simultaneously contain the four genes required to control the ErbB pathway. miRBase, Tar-getScan, MiRanda, PicTar, miRWalk, RNA22, PITA and MicroCosm Targets were used to find a suitable miRNA and reducing experimental error and trials. The results were checked out with miRWalk, TargetScan, RNA22 and DIANA mirPath software programs and the one, which was most effective on ABL2, PAK2, EGFR and MAPK1, was selected.

Cell culture
The human lung cancer cell lines (A549 and Calu-6) were obtained from National Cell Bank of Iran (Pasteur Institute of Iran, Tehran, IRAN) and grown in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum (Thermo Fisher Scientific) and antibiotics (100 units/mL of penicillin and 100 μg/mL of streptomycin). The cells were maintained at 37 °C in a humidified atmosphere of 5% CO 2 .

Clinical specimens
All thirty-five lung cancer tissue samples and nine nontumor tissue samples (0.5 cm to 2 cm distance from the tumor), as control were obtained from the Imam Khomeini Hospital Complex (Tehran, Iran). The present study was conducted under the instructions accepted by the Ethics Committee of Pasteur Institute of Iran, and written informed consent was obtained from all participants involved in the present study. The tissue samples were stored in liquid nitrogen until analysis. All experiments and analysis procedures were performed in accordance with the relevant guidelines and regulations.

Transfection
Pre-miR precursors of miRNA-377 and control pre-miR precursor (scrambled) were purchased from Ambion (Thermo Fisher Scientific). A549 and Calu-6 cells were grown to 80% confluence in 6-well plate at the density of 5 × 10 5 per well one day before transfection, cells were transfected with miRNA-377 or control scrambled oligonucleotide (100 nM of the final concentration of mixture) with Lipofectamine 2000 (Thermo Fisher Scientific) according to the procedures described in the manufacturer's protocol. Cells were harvested after 24, 48 and 72 h for subsequent analysis.

Luciferase assay
To test the direct binding of miRNA-377 to the target genes ABL2, PAK2, EGFR and MAPK1, a luciferase reporter assay was done. For luciferase reporter experiments, 3′-UTR of target genes ABL2, PAK2, EGFR, MAPK1 and a special scrambled sequence (AAG CTT CAT AAG GCG CAT AGC) as a negative control were cloned into psiCHECK™-2 Vector (Promega, C8021) closely downstream to the stop codon of Renilla luciferase (The PCR primers are listed in Table 3 Supplementary data). Luciferase assays were performed with Dual-Glo Luciferase Assay System (Promega, E2940). In order to perform this test, 4-5 × 10 4 of A549 and Calu-6 cells were cultured into each well of a 48-well plate 1 day before transfection. The cells were transfected with 400 ng psiCHECK™-2 Vector harboring, ABL2 3′-UTR, PAK2 3′-UTR, EGFR 3′-UTR MAPK1 3′-UTR or scrambled sequence and 100 nM of each precursor miRNAs (377 or scrambled miRNAs) using Lipofectamine 2000 reagent (Life Technology) according to the manufacturer protocol. The multi-well plate luminometer Renilla luciferase activity was normalized to that of firefly luciferase. Luciferase activities were determined 24 h after transfection using dual luciferase reporter assay system (Promega) [23].

Designing the primers and stem-loop
The sequence of the miRNA-377 was retrieved from the miRBase at http:// www. mirba se. org/. In order to increase the flexibility of stem-loop structure as well as achieving necessary sensitivity, the published sequence of Chen et al. was modified [24]. The modifications included addition of fourteen nucleotides to the original sequence in order to lengthen the loop and enabling the design of a universal reverse primer inside it and substitutions for decreasing the melting temperature of the stem part. This new designed structure was able to specifically detect each miRNA because few nucleotides complimentary to the 3'UTR of miRNA were added to each stem-loop [25]. Almost complete sequence of miRNA-377 was used as the forward primer for real-time PCR assay. miRNA forward primer and the mRNA real-time PCR primers were designed with Allel ID, Gene Runner software programs and Primer blast (Table 4 Supplementary data) were designed by Allele ID6 software (PREMIER Biosoft, USA). Secondary structure analysis of the amplicon was performed with ''mfold'' software (http:// mfold. rna. albany. edu/?q= mfold/). All Oligos were purchased from Metabion international AG (Lena-Christ-Strasse, Germany).

RNA isolations and quantitative real-time PCR
miRNeasy Mini kit (Qiagen) was used to extract total RNA from both the cells and tissues. The concentration and purity of total RNA were quantified using UV spectrophotometric analysis at 260 nm. The cDNA synthesis kit (Thermo Fisher Scientific) was used to synthesize cDNA of mRNA. Real-time PCR was carried out using real-time PCR Master Mix (2× Maxima SYBR Green real-time PCR Master Mix, Thermo Fisher Scientific) and the GAPDH was chosen as the endogenous control. The PCR mixtures were made in 25 μL volumes consisted of 12.5 μL of SYBR Green real-time PCR Master Mix, 2 μL of cDNA, 0.4 μL of Forward Primer (10 pmol), 0.4 μL of Reverse Primer (10 pmol) and 7.2 μL of RNase-free H 2 O. The thermal cycling profile of real-time PCR was as follows: 10 min initial denaturation at 95 °C, followed by 40 cycles of 95 °C for 10 s, 60 °C for 40 s. Finally, the melting curve analysis was performed at a range of 65 °C up to 95 °C. For miRNA following RNA extraction, the samples were reverse transcribed using M-MuLV reverse transcriptase enzyme. Five microliters of extracted miRNA, was added to 1.5 μL of stem-loop RT primers (50 nM) and 4 μL distilled water. The 10.5 μL reactions were incubated in a peQlab thermocycler (peQlab, Germany) at 65 °C for 10 min. The tubes were hold on ice and 4 μL of 5× RT-Buffer (Roche, Germany), 2 μL of DTT (10 mM) (Merck Germany), 2 μL of dNTP (10 mM), 20unit of RNase inhibitor (Thermo scientific, USA) and 1 μL of by M-MuLV reverse transcriptase enzyme were added to the preheated mixture. The cDNA synthesis was carried out as following: 25 °C for 15 min, 37 °C for 15 min, 42 °C for 40 min and finally, 2 min' incubation at 95 °C in order to inactivate the enzyme. The cDNA was stored at − 20 °C until use.
Real-time PCR for miRNA-377 was performed in 12.5 μL PCR mixture volume composed of 6.5 μL 2× Maxima qPCR Master Mix (Takara, Japan), 0.3 μM of each oligonucleotide primers and 2 μL of cDNA. Amplification was performed in the following conditions: Enzyme activation step at 95 °C for 10 min and 45 cycles of two thermal amplification steps: 94 °C for 10 s and 60 °C for 40 s. Single fluorescence detection was performed in each cycle at the end of extension phase. Reaction set ups were done manually. Amplification, data acquisition and analysis were performed on Rotor-Gene 6000 (Corbett Research, Australia) using Rotor-Gene 6.1 and the Relative expression software tool (REST). All samples were run in triplicate along with pertaining controls such as positive, negative controls. U6 was used as the reference gene. The Ct is defined as the fractional cycle number at which the fluorescence passes the fixed threshold. Permissible Ct scattering in triplicates of each sample was ≤ 5%. Statistical analysis for relative miRNA and mRNA expression were performed by REST proposed by Pfaffl. Fold change and P value (≤ 0.05) were estimated by REST, where all requirements of this software were considered.

Apoptosis
Cells were seeded into 12-well plates at a concentration of 3 × 10 4 cells/well and allowed to adhere to the surface in 24 h. After 48 h of transfection, cells were trypsinized, placed onto 1.5 mL tube. After washing and centrifugation, the harvested cells were incubated in PI and Annexin V FITC according to the manufacturer's protocol (Roche Applied Sciences). Stained cells were analyzed using a flow cytometer (Partec, Germany). In each experiment, 20,000 cells per 200 s were recorded and each sample was run in triplicate.

Cell proliferation
MTT (Sigma) was used to perform cell proliferation assay. Cells were seeded into 96-well plates at a concentration of 1 × 10 4 cells/well. After 72 h of transfection, cells were washed with PBS and MTT (5 mg/mL) added. The cells were incubated for 3 h at 37 °C in the CO 2 incubator. The crystals were then dissolved by adding 100 μL of DMSO. Spectrophotometrical absorbance of the purpleblue formazan dye was measured in a microplate reader at 540 nm (reference wavelength: 630 nm). Each experiment was repeated at least three times.

Statistical analysis
Real-time PCR experiments, cell proliferation and apoptosis assays were carried out atleast three times. The data shown are the mean ± standard error of the mean. Statistical analysis of data was carried out using the Prism 8 (GraphPad) software using a Student's t test for the comparison of the difference between the two groups and analysis of variance (ANOVA) followed by post hoc Tukey's Multiple Comparison Test, for comparison among multiple groups. All real-time PCR results were analyzed using ∆∆CT method with REST 2009 software (Qiagen, Hilden, Germany) and expression was normalized against GAPDH for mRNA and U6 for miRNA. Correlation analysis was performed using the Spearman test. The differences were considered statistically significant at P values less than 0.05.

Demographic data of patients
While twenty-two of samples were obtained from men, thirteen samples were from women. The age ranges of patients were 39-80. They all suffered from adenocarcinoma of the lung, TNM stages in patients were T1N1Mx in seven patients, T2N2M1 in thirteen patients, T3N0M0 in ten patients and T4N2M0 in five patients and EGFR mutation had been detected in three patients.

miRNA-377 can target the 3ʹ-UTR of EGFR, PAK2, MAPK1
Eight bioinformatics prediction software programs (miR-Base, TargetScan, MiRanda, PicTar, miRWalk, RNA22, PITA and MicroCosm Targets) were applied to predict the best miRNA for silencing ABL2, PAK2, EGFR and MAPK1 genes in ErbB pathway. Four first ranked miRNAs for each gene are shown in Table 1 (Supplementary data). hsa-miRNA-377-3p was among the first four miRNAs that could recognize and attach to all four mRNAs. In order to confirm bioinformatics prediction of previous software programs and predict the miRNA-377-target positions interaction and seed matching with ABL2, PAK2, EGFR and MAPK1, prophecy programs including miRWalk, Tar-getScan, RNA22, DIANA, mirPath, miRmap and miRDIP software programs were used (Table 1). Inhibitory effect of miRNA-377 on all four genes expression was predicted at least in four prediction software programs with moderate to high score.

Expression level of miRNA-377 in lung cancer tissues and lung cancer cell lines was lower than normal tissues
To investigate the level of miRNA-377 in lung cancer samples, A549 and Calu-6 cell lines and normal tissues, total RNA were extracted from thirty-five NSCLC patients and nine normal samples used for real-time PCR. The results are shown that the average miRNA-377 expression in human NSCLC tissues and cell lines was significantly (P < 0.05) lower than that found in non-tumor tissue samples. We compare them due to the fact that we do not have normal cell line (Fig. 1b and c).

There was a negative correlation between the expression of miRNA-377 and some ErbB signaling pathway genes mRNA level in lung cancer tissues
In order to confirm any correlation between the level of miRNA-377 and EGFR, MAPK1, and PAK2 mRNA expression in lung cancer samples and normal tissues, total RNA was extracted from thirty-five NSCLC patients and nine normal samples used for real-time PCR.
The EGFR, MAPK1 and PAK2 mRNAs were significantly increased in the lung tumor tissues compared to normal tissues. (P < 0.05; Fig. 2a).

miRNA-377 introduction to A549 and Calu-6 cells downregulates the expression level of some genes belonging to ErbB signaling pathway
To determine whether miRNA-377 could target the EGFR, ABL2, MAPK1 and PAK2 mRNAs, we examined the relative expression levels of the target RNAs in transfected A549 and Calu-6 cell lines with miRNA-377 or scrambled oligonucleotide and evaluated with control cells by real-time PCR. In this experiment non-transfected cells and cells transfected with scrambled oligonucleotide were used as controls, respectively. The real-time PCR results demonstrated that miRNA-377 induced a remarkable decrease in levels of EGFR, MAPK1 and PAK2 mRNAs in the two cell lines (Fig. 3). The suppression effect of miRNA-377 on ABL2 levels in A549 and Calu-6 cells was not significant.

Overexpression of miRNA-377 induced apoptosis in Lung cancer cell lines
The apoptotic effect of miRNA-377 in A549 and Calu-6 cells was evaluated by flow cytometry using annexin V and PI. The results obtained demonstrated an increase in cells undergoing early apoptosis when compared controls (Fig. 4a  and 4b). Non-transfected cells and cells transfected with scrambled oligonucleotide used as controls for this experiment. The apoptosis ratio significantly increased in cells transfected with miRNA-377 in comparison with controls (P < 0.05). The percentage of early apoptotic cells increased from 0.16% to 30.41% in A549 and 0.2 to 24% in Calu-6 transfected with miRNA-377. In order to more information about the effect of miR-377 on apoptosis induction and based on the result of other study [26], the expression of X-linked Inhibitor of Apoptosis Protein (XIAP) as a valid Results are shown as mean ± SE from three independent experiments, which performed in triplicates. *P < 0.05 compared with control target of miR-377 in transfected A549 and Calu6 was evaluated. The real-time PCR results demonstrated that miRNA-377 induced a remarkable decrease (P < 0.05) in levels of XIAP in the two cell lines (Fig. 4c).

Overexpression of miRNA-377 decreased cell proliferation in lung cancer cell lines
In order to understand the role of miRNA-377 on lung cancer cell lines cancer cell proliferation, MTT assay was used. A549 and Calu-6 cells were seeded in 96-well plates and transfected with miRNA-377 or scrambled oligonucleotide. 48 h after transfecting, the proliferation of these cells in comparison with non-transfected and scrambled cells was evaluated. We demonstrated that the miRNA-377 could significantly (P < 0.05) reduce cell growth compared with the scrambled and control groups in the A549 and Calu-6 cell lines (Fig. 4d). Experiments were performed three times.

Discussion
Lung cancer is the leading cause of cancer-related deaths in many countries [27]. The ErbB pathway plays an important role in the pathogenesis of many types of human cancer, including those of the lung [9]. Accumulating evidence suggested that some miRNAs are abnormally expressed in several types of cancer [28,29]. In this article, we studied the role of miRNA-377 in ErbB pathway in lung cancer and found the correlation of the downregulation of miRNA-377 and overexpression of three genes in the ErbB pathway in NSCLC tissue. In addition, we demonstrated The results showed that miRNA-377 could markedly inhibit cancer cell proliferation that miRNA-377 significantly increased the apoptosis and decreased cell proliferation of lung cancer cell lines in vitro. The interaction between miRNA-377 and 3ʹ-UTR of the mRNA of EGFR, MAPK1, ABL2 and PAK2 was investigated through in-silico analysis. Among all of the miRNAs that targeted the 3ʹ-UTR of mentioned mRNAs, miRNA-377 had a relatively high score for assessment in the present. Also, luciferase reporter data showed that 3′-UTR of PAK2, EGFR or MAPK1 are functional target site for miRNA-377 in A549 and Calu-6 cells. miRNA-377 is one of the miR-NAs which belongs to the large miRNA cluster encoded on chromosome 14 (14q32) and plays critical roles in types of human malignancies [29,30]. The role of miRNA-377 downregulation was identified to be associated with various human malignancies, including prostate tumors, hepatocellular carcinoma, osteosarcoma MG-63, clear cell renal cell carcinoma, glioblastoma, malignant melanoma, pancreatic cancer and ovarian cancer [31][32][33][34][35][36][37]. Also, miRNA-377 plays an important role in chemotherapeutic resistance in B-cell lymphoid malignancies [38]. One study has been shown that miRNA-377 functions as an oncogene in human colorectal cancer [39]. Previously, we have identified the role of miRNA-377 in the regulation of DNA methylation in pancreatic cancer [40]. Decreasing of miRNA-377 has been found in lung cancer [41] and inhibition of tumorous behaviors of miRNA-377 in this cancer through targeting AEG-1 and CDK6, has been reported [41,42].
In this study, we found significant diminution of miRNA-377 and overexpression of EGFR, MAPK1 and PAK2 genes in NSCLC tissue samples in comparison to their control. Real-time PCR was used to evaluate the level of miRNA-377 in both lung tumor tissue and lung cancer cell lines compared to their normal samples. The data showed that miRNA-377 was significantly downregulated in lung cancer samples compared to normal tissues and cells (Fig. 1). To evaluate the levels of EGFR, MAPK1, and PAK2 expression, we analyzed their mRNA levels in all NSCLC and normal samples (Fig. 2). Our results showed that mRNAs of EGFR, MAPK1 and PAK2 were significantly upregulated in tumors compared to normal tissues.
The data is confirming the inverse correlation between miRNA-377 and EGFR, MAPK1 and PAK2 mRNAs. Expression of miRNA-377 was decreased while mRNA of EGFR, MAPK1 and PAK2, the ErbB signaling pathwayrelated genes were increased in NSCLC tissue when compared with the control group.
To confirm whether miRNA-377 could target EGFR, MAPK1, ABL2 and PAK2 mRNAs in A549 and Calu-6 cells, we analyzed expression levels of them in these cells, following miRNA-377 transfection. The miRNA-377 at the same time reduced the expression level of mRNA of EGFR, MAPK1 and PAK2 genes. While no significant effect was detected with the expression levels of ABL2 mRNA (Fig. 3).
An MTT assay and flow cytometry were used to determine the roles of miRNA-377 in cell proliferation and apoptosis, respectively. The results indicated that, compared with the control and scrambled miRNA, miRNA-377 obviously inhibited the cell proliferation and promoted the apoptosis of A549 and Calu-6 cells, which suggested that miRNA-377 has a role in suppressing lung cancer. These results were in line with previous studies, which reported that of miRNA-377 suppresses prostate cancer, hepatocellular carcinoma, human osteosarcoma, clear cell renal cell carcinoma, glioblastoma, pancreatic cancer, ovarian cancer cells proliferation, and promotes prostate cancer, pancreatic cancer cells and osteosarcoma apoptosis [26,30,[32][33][34][35][36][37].
In osteosarcoma miR-377 induced apoptosis by targeting XIAP. This protein can concurrently prevent the expression of Caspases-9 in the key proteases Caspases-3, Caspases-7, and mitochondria-dependent apoptotic pathways [26]. Furthermore, another study showed that miR-377 suppressed BCL-xL expression. This protein is a member of the Bcl-2 family of proteins, and acts as an anti-apoptotic agent [38].
All data indicated that miRNA-377 has a key role in cell proliferation and apoptosis of lung and mentioned cancer cells. The corresponding molecules involved in the mentioned processes and more miRNA-377 targets remain to be clarified.

Conclusions
Taken together, we found the role of miRNA-377 in the regulation of ErbB pathway in lung tumor via targeting the 3ʹ-UTR of the mRNA of EGFR, MAPK1, and PAK2. Transfection of miRNA-377 causes preventing cell proliferation and promoting cell apoptosis in A549 and Calu-6 cells. Future studies will maybe find further miRNA-377 targets to clarify a complete picture of the miRNA-377 regulation network that drives the progression of NSCLC. These data also indicate that miRNA-377 may be a potential therapeutic target for the treatment of NSCLC.
Data availability The authors confirm that the data supporting the findings of this study are available.

Declarations
Conflict of interest All authors declare that they have no conflict of interest.
Ethical approval The present study was conducted under the instructions accepted by the Ethics Committee of Pasteur Institute of Iran, written informed consent to participate, and consent to publish forms was obtained from all participants involved in the present study. This material is the authors' own original work, which has not been previously published elsewhere. The paper is not currently being considered for publication elsewhere. The paper reflects the authors' own research and analysis in a truthful and complete manner.