MicroRNA‐130b targets PTEN to induce resistance to cisplatin in lung cancer cells by activating Wnt/β‐catenin pathway

More and more studies indicate the relevance of miRNAs in inducing certain drug resistance. Our study aimed to investigate whether microRNA‐130b‐3p (miR‐130b) mediates the chemoresistance as well as proliferation of lung cancer (LC) cells. MTS assay and apoptosis analysis were conducted to determine cell proliferation and apoptosis, respectively. Binding sites were identified using a luciferase reporter system, whereas mRNA and protein expression of target genes was determined by RT‐PCR and immunoblot, respectively. Mouse xenograft model was used to evaluate the role of miR‐130b in cisplatin resistance in vivo. The rising level of miR‐130b in cisplatin resistance LC cell lines (A549/CR and H446/CR) versus its parental cell lines, indicated its crucial relevance for LC biology. We identified PTEN as miR‐130b's major target and inversely correlated with miR‐130b expression in LC. Moreover, excessive miR‐130b expression promoted drug resistance and proliferation, decreased apoptosis of A549 cells. Suppression of miR‐130b enhanced drug cytotoxicity and reduced proliferation of A549/CR cells both internally and externally. Particularly, miR‐130b mediated Wnt/β‐catenin signalling pathway activities, chemoresistance and proliferation in LC cell, which was partially blocked following knockdown of PTEN. These findings suggest that miR‐130b targets PTEN to mediate chemoresistance, proliferation, and apoptosis via Wnt/β‐catenin pathway. The rising level of miR‐130b in cisplatin resistance LC cell lines (A549/CR and H446/CR) versus its parental cell lines, indicated its crucial relevance for LC biology. Moreover, excessive miR‐130b expression promoted drug resistance and proliferation, decreased apoptosis of A549 cells. These findings suggest that miR‐130b targets PTEN to mediate chemoresistance, proliferation, and apoptosis via Wnt/β‐catenin pathway.

the underlying mechanisms of chemoresistance. 5 A number of studies explored cisplatin, an efficient spectrum drug against cancer that is frequently applied in the treatment of various cancers in the place of lung, breast, bladder and brain, etc. 6,7 Cisplatin triggers cancer cell death by cross-linking with the DNAs to suppress replication and transcription. 8 However, extended records of administrating cisplatin caused great drug fastness in those cisplatin-applied tumour cells. 9,10 In order to keep the effectiveness of the cisplatin treatment, it is imperative for lung cancer cells to maintain a steady level of sensitivity against it.
Considering recent studies that demonstrated the correlation between cancer cells and resistance to cisplatin, we examined how miR-130b affects the cisplatin-resistance in lung tumour cells in our research.
MicroRNAs (miRNAs) are non-coding RNA molecules with around 20 to 25 nucleotides that can lead to a downregulation of target proteins through the degradation of this mRNA or through translational inhibition, which play an important role in various malignancies. 11,12 Abnormal miRNA expression has been observed in both physiological and pathological processes multiple human cancers like proliferation, invasion, apoptosis, and chemotherapy resistance. 12 MicroRNA-130b-3p (miR-130b) targets CYLD to suppress growth of cells and induce programmed death in human gastric cancer cells. 13,14 Moreover, miR-130b was recorded to be lifted in triple negative breast cancer tissue in comparison with adjacent healthy ones, and miR-130b mediated CCNG2 that could be closely connected with the deteriorating development of the cancer in question. 15 However, the role of miR-130b in chemoresistance lung cancer cells is still unknown.
In this study, we aimed to explore the role of miR-130b in cisplatinresistance lung cancer cells. The upregulation of miR-130b was identified in cisplatin-resistance lung cancer cells. We found that miR-130b responds to cisplatin resistance through altering the targeted PTEN level and subsequence Wnt/β-catenin pathway. The discovery of miR-130b/ PTEN being a new regulator that controls cisplatin-resistance in lung cancer offers a fresh molecular insight that might be utilized in new therapy development for cisplatin resistance in lung cancer.

| Cultured cells and chemical reagents
Our study adopts the cell lines A549 and H446 from the American Type Culture Collection in Manassas. The cisplatin-resistant A549/CR and H446/CR cells were derived by incubation with stepwise increasing cisplatin concentrations. The cells were routinely cultured in RPMI-1640 medium plus 10% fetal bovine serum (Gibco, NY) in humidified 5% CO 2 incubator with temperature of 37°C. Cisplatin was obtained from selleckchem. MiR-130b inhibitor, miR-130b mimic (miR-130bm), or the appropriate negative controls (NC) of miRNA inhibitor (miR-iNC) and miRNA mimic (miR-NC) were brought from GenePharma (Shanghai, China).

| Cell viability
Cells for experiments were cultured overnight in plates with 96 wells (4 × 10 3 cells/well). Subsequently, MTS assay was carried out with Promega MTS assay kit operated in accordance with manufacturer's instructions. The Wallac Victor 1420 Multilabel Counter from Perkin-Elmer was used to determine luminescence measure. Each assessment was done in triplicate with 3-time repetition to ensure minimum deviation.

| SIRNA AND TRANSFECTION
PTEN siRNA and control siRNA were obtained from Santa Cruz. A549 and H446 cells had been seeded in plates with 12 wells for exactly 1 day to reach 20% to 30% confluence and then gone through transfection with Lipofectamine 2000according to manufacturer Invitrogen's manual. PTEN plasmid was obtained from Addgene. The above cells in plates with 30% to 40% confluence after 24-hour cultivation were transfected with PTEN or NC plasmid using Lipofectamine 2000 abiding by the producer (Invitrogen)'s instructions. To determine the influence of miR-130b on chemosensitivity and programmed death of cells, lung cancer cells went through transfection with MiR-130b inhibiter or miR-130b mimic.

| RNA extraction and qRT-PCR
After transfection for 24 hours, the cells' total RNA was obtained with Trizol (Invitrogen, Carlsbad, CA) following producers' manual. The synthesis of cDNA was carried out with the RevertAid™ First Strand cDNA Synthesis Kit of Thermo Fisher Scientific Inc. The qRT-PCR was carried out with Takara's SYBR Premix Ex Taq II kit in the Bio-Rad's CFX96 real-time PCR system. The RNA levels of target genes were standardized with that of β-actin gene by the 2 −△△Ct way. Each reaction was conducted in triplicate and with 3-time repetition to ensure minimum deviation.

| Apoptosis analysis
Hoechst 33258 nucleic acid stain purchased from Invitrogen was used for nuclear staining for the analysis of apoptosis. 16,17 Annexin V/ propidium iodide (PI) staining was conducted with annexin-Alexa 488 (Invitrogen) and PI. Caspase activity was evaluated via the SensoLyte Homogeneous AMC Caspase-3/7 Assay Kit (Anaspec).

| Luciferase reporter analysis
To determine the impact of miR-130b, the 3′ UTR of PTEN was mag-

| Xenografts
The experiments related to animals were conducted under the Guide cells/mL and administrated into the side of BALB/C female athymic mouse aged 5 to 6 weeks (n = 6). Cisplatin (3 mg/kg), polyplex containing 500 nM miR-21 inhibitor in 50 μL of PBS, or their combination was applied via intraperitoneal injection twice a week. The development of tumour was closely monitored by callipers, and its volumes were calculated with the formula of 1/2 × length × width. 2 Mice were euthanized once tumours have grown into~1.0 cm 3 or larger. After dissection, tumour sections were put in 10% formalin and embedded in paraffin, which then went through TUNEL immunostaining with an AlexaFluor 488-conjugated secondary antibody (Invitrogen) to detect the signals.

| Statistical analysis
The analysis was conducted with GraphPad Prism V (Graphpad Software, CA, USA), and all figures were recorded as means ± SD. A t-test was used to compare among various groups. P < 0.05 was viewed as statistically significant.

| Cisplatin-resistant A549 and H446 cell generation
For exploring the mechanism of cisplatin-resistance in lung cancer, we exposed A549 and H446 cells to cisplatin with rising levels of concentration for over 1 year. The cells that survived from each session with >70% cell confluency were passed through trypsinization, which leads to an elevated level of cisplatin concentration. The process was   Figure 3A). In comparison with the NC group, the A549 and H446 cells that transfected with miR-130b mimic are more resistant to cisplatin ( Figure 3B). The cisplatin-induced apoptosis rate of miR-130bm-transfected A549 and H446 cells was obviously much lower than that of the NC group ( Figure 3C). For continuous exploration on the influence of miR-130b on cisplatin-induced apoptosis in lung cancer, miR-130 inhibitor was transfected with A549 and H446 cells. qRT-PCR showed that the miR-130b inhibitor downregulated miR-130b expression of these cells ( Figure 3D). Inhibition of miR-130b significantly enhanced cells' sensitivity towards cisplatin ( Figure 3E). All above results suggested that miR-130b could facilitate lung cancer cells' resistance towards cisplatin.

| MiR-130b mediates PTEN protein expression via targeting 3′UTR in lung cancer
PTEN gene was regarded as a target of miR-130b (Target Scan, http:// www.targetscan.org) ( Figure 4A). To prove this, a luciferase reporter gene vector was built with the downstream target luciferase gene,

| Overexpression of miR-130b activates Wnt/βcatenin pathway through PTEN inhibition
In order to determine the function of PTEN on cisplatin resistance mediated by miR-130b, PTEN plasmid or control vector was processed into the miR-130b mimic-transfected cisplatin-resistant A549 cells to measure the cell viability in different cisplatin concentration conditions. Western blotting analysis indicated that PTEN overexpression greatly upregulated PTEN's protein level ( Figure 5A) and also considerably decreased the survival rate of the A549 cisplatin resistance cells than in the NC group ( Figure 5B). This indicated that miR-130b may regulate cisplatin resistance in the A549 cisplatin resistance cells via reducing PTEN protein level.
In addition, Western blotting analysis demonstrated that the miR-130b overexpression reduced the PTEN expression and decreased the phosphorylation of downstream kinase β-catenin ( Figure 5C). PTEN has the ability of suppressing various types of tumours and is a negative regulating factor of Wnt/β-catenin. 18 Hence, we assumed that miR-130b's overexpression could activate Wnt/β-catenin pathway via cutting down PTEN's protein level, which led to A549 cells' resistance to cisplatin. For further exploration of the impact of Wnt/ β-catenin pathway on miR-130b-induced cell survival and cisplatin resistance, we treated the miR-130b mimic transfected A549 or A549/PTEN cells with cisplatin in different dosages. Findings demonstrated that PTEN abrogated partly the phosphorylation of β-catenin triggered by miR-130b transfection ( Figure 5D). Meanwhile, miR-130b-induced cisplatin resistance was greatly suppressed ( Figure 5E).
All the above indicated that miR-130b overexpression was able to activate the Wnt/β-catenin pathway through downregulating PTEN in lung cancer cells.

| Targeting miR-130b overcome cisplatin resistance in vivo
To evaluate the potential function of miR-130b on cisplatin therapy internally, A549 cells with overexpressed miR-130b were implanted in mice. The miR-130b levels in the miR-130b-stable A549 cells compared with that in the routine A549 were demonstrated in Figure 6A.
We discovered that the average size of tumours in the miR-130b group was considerably larger than the EV group even though they were administrated with the same dosage of cisplatin ( Figure 6B). This showed the overexpression of miR-130b resistance in the anti-tumour effect of cisplatin within the body. We also examined tumour cells apoptosis in xenografts, which demonstrated that cisplatin-triggered cell apoptosis was reduced while miR-130b was overexpressed (  inhibitor in lung cancer. Tumour cell death was also aligned with the same trend ( Figure 6E). The results indicated that targeting miR-130b may assist to overcoming cisplatin resistance.

| DISCUSSION
The general cause of drug resistance in tumour cells is due to accumulation of changes in genes altered the genetic pathways, thus stimulating cancer cell evolution of phenotypes. 19,20 There have been limited effective solutions to solve the resistance problem in cancer treatment over the years. 19,21 Previous researches have demonstrated that PTEN is related with the drug resistance in various cancers. 22 Our study gave evidence to the mechanistic connection of miR-130b upregulation in cisplatin resistance in lung cancer.
Our findings suggest that miR-130b targets PTEN to mediate chemoresistance, proliferation, and apoptosis via Wnt/β-catenin pathway in lung cancer cells.
Lately, miRNAs have been shown as a crucial regulator for the cellular responses of tumour cells against treatment. 23 Patients' response to chemotherapy has been proved to connect tightly with the functioning of miRNAs. 24,25 Although its underlying mechanism still remains unsure, experiments and observations have so far revealed several possibilities of roles that miRNAs could play, such as changing the targets of certain drug, affecting therapy-induced apoptosis, mediating proteins involved in multiple drug resistance, and stimulating angiogenesis activities, etc. In our research, we discovered that miR-130b upregulated A549/CR cells in comparison with the A549 cells experiment group. The findings revealed that overexpression of miR-130b encouraged resistance to cisplatin in lung cancer cells, and the inhibition of miR-130b overexpression could reverse cisplatin FIGURE 5 miR-130b promotes cisplatin resistance via Wnt/β-catenin signalling pathway. (A) A549 cells transfected with miR-130b mimic with or without PTEN plasmid co-transfection, PTEN expression were analysed by western blotting. (B) A549 cells transfected with miR-130b mimic with or without PTEN plasmid co-transfection were treated with increasing concentrations of cisplatin for 72 hours. Cell proliferation was determined by MTS assay. (C) A549 cells were transfected with miR-130b mimic or mimic-NC for 24 hours, indicated protein level was analysed by western blotting. (D) A549 and A549/PTEN cells were transfected with miR-130b mimic or mimic-NC for 24 hours, indicated protein level was analysed by western blotting. (E) A549 and A549/PTEN cells were transfected with miR-130b mimic or mimic-NC for 24 hours and then treated with 40 μM of cisplatin for 24 hours resistance. PTEN was viewed to be 1 target of miR-130b, and its signalling pathway has been recorded to regulate a variety of cellular activities in tumour settings including programmed cell death, cell proliferation, invasion, etc. In this sense, the hypothesis of miR-130b targeting PTEN is in accordance with its acknowledged biological impact. Actually, certain miRNAs can assist resistance via targeting PTEN in different tumours.
PTEN loss is a commonly seen genetic variation in cancers like, gastric cancer, breast cancer, and spongioblastoma. 26,27 It is closely connected with cytotoxic drug resistance. Our study suggested that miR-130b mediated the gene expression of PTEN in lung cancer cells with a negatively correlation. Consequently, the aggressiveness of these cells was intensified after the miR-130b expression transfection process. Increasing evidences demonstrated that PTEN malfunction had prognostic implications in some malignant tumours like lung cancer. Our research findings showed that targeting PTEN at posttranscriptional stage via miRNAs, eg, miR-130b could regulate its downregulation process. All the findings indicated that elevated miR-130b expression could be related with diminishing survival rate of lung cancer patients and the miR-130b inhibitor bears possible capability of countering drug resistance. Reducing miR-130b's level may effectively increase the sensitivity of tumour cells to cisplatin. We expect treatment strategy to be developed based on miR-130b levels. Combination cisplatin with the inhibition of miR-130b could enhance the sensitivity and effectiveness of drugs in lung cancer. In addition, we revealed that miR-130b reacts to cisplatin resistance in lung cancer cells via altering its target PTEN's level and downstream Wnt/βcatenin signalling pathway. Wnt/β-catenin signal pathway is among the most crucial signalling pathways during embryonic development and remains a popular problem for tumour research. 28 Recently, accumulating evidence reported the relevance of Wnt/β-catenin signal pathway in the chemoresistance of various cancers. 29 Hereby, we revealed that Wnt/β-catenin has a key influence on miR-130b mediated cisplatin resistance.

| CONCLUSIONS
Our results uncovered the unique function of miR-130b in cisplatinresistance, which was done mainly through activating Wnt/β-catenin signalling pathway of lung tumour. Although the resistance to drug is a main problem for the lung cancer therapy involved cisplatin, the method for inhibition of the miR-130b could be a novel way to improve the sensitivity of lung cancer to cisplatin.