A positive feedback loop between miR‐181b and STAT3 that affects Warburg effect in colon cancer via regulating PIAS3 expression

Abstract This study aimed to investigate the relationship between the expression of microRNA (miR)‐181b, protein inhibitor of activated STAT3 (PIAS3) and STAT3, and to examine the function of the miR‐181b/PIAS3/STAT3 axis on the Warburg effect and xenograft tumour growth of colon cancer. Moreover, a positive feedback loop between miR‐181b and STAT3 that regulated the Warburg effect in colon cancer was explored. A luciferase reporter assay was used to identify whether PIAS3 was a direct target of miR‐181b. The gain‐of‐function and loss‐of‐function experiments were performed on HCT 116 cells to investigate the effect of miR‐181b/PIAS3/STAT3 on the Warburg effect and xenograft tumour growth of colon cancer, as determined by commercial kits and xenograft experiments. The relationship between the expression of miR‐181b, PIAS3 and STAT3 in HCT 116 and HT‐29 cells was determined using RT‐qPCR and Western blot. We found miR‐181b was a direct regulator of PIAS3. miR‐181b promoted the Warburg effect and the growth of colon cancer xenografts; however, these effects could be reversed by PIAS3. miR‐181b expression interacted with STAT3 phosphorylation in a positive feedback loop in colon cancer cells via regulating PIAS3 expression. In conclusion, this study for the first time demonstrated that miR‐181b contributed to the Warburg effect and xenograft tumour growth of colon cancer by targeting PIAS3. Moreover, a positive feedback loop between miR‐181b and STAT3 that regulated the Warburg effect in colon cancer was also demonstrated. This study suggested miR‐181b/PIAS3/STAT3 axis as a novel target for colon cancer treatment.


| INTRODUCTION
Colon cancer is a leading cause of cancer death in the worldwide. 1,2 About 1 million people suffer colon cancer every year, and it causes 0.6 million deaths annually around the world. 3 Despite advances in surgical treatment, the 5-year overall survival rate of patients with metastatic colon cancer is only 5%-10%. 4 Therefore, it is necessary to investigate the molecular mechanisms underlying colon cancer development so as to contrive novel strategies for colon cancer treatment.
Cancer cells rewire their metabolism to promote cancer progression. The common feature of this altered metabolism is that cancer cells preferentially produce energy by a high rate of glycolysis followed by lactic acid fermentation in the cytosol, 5,6 rather than by a comparatively low rate of glycolysis followed by oxidation of pyruvate in mitochondria as in most normal cells. 7 This phenomenon is referred as the Warburg effect, which has been documented for over 90 years. The Warburg effect is considered as one of the most fundamental metabolic alteration associated with malignant transformation; however, it remains unclear how the Warburg effect is regulated during tumour progression.
MicroRNAs (miRNAs or miR) are a class of small non-coding RNAs that regulate gene expression at the post-transcriptional and translational levels through binding to the 3′untranslated region (3′UTR) of target mRNAs. The roles of miRNAs in cancer progression have been well-documented and several miRNAs such as miR-223, miR-133, and miR-200 family have been implicated in the regulation of aerobic glycolysis in cancer. 8 MiR-181b is an important miRNA which is correlated with tumorigenesis. 9 MiR-181b has been shown to exert its effect on cancer by regulating cell proliferation, apoptosis, invasion and migration. 10,11 Recently, the role of miR-181b in regulating aerobic glycolysis in cancer has attracted much concern. 12,13 Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that mediates cellular responses to various cytokines and growth factors. Recently, some studies reveal that STAT3 acts as a master regulator of aerobic glycolysis. [14][15][16] regulates the Warburg effect via promoting aerobic glycolysis and downregulating mitochondrial activity. 16 Protein inhibitor of activated STAT 3 (PIAS3) is a specific inhibitor of STAT3. It has been shown that ectopic expression of PIAS3 in cancer cells can suppress the transcriptional activity of STAT3 and inhibit tumour growth. 17,18 In the present study, we explored the relationship between miR-181b (miR-181b-5p), PIAS3 and STAT3, and investigated the function of miR-181b/PIAS3/STAT3 axis on the Warburg effect and xenograft tumour growth of colon cancer. Moreover, a miR-181b-STAT3 positive feedback loop that contributed to the Warburg effect in colon cancer cells was demonstrated as well.
The sequences of miRNAs and siRs were shown in Table 1. Cell transfection (plasmid 4.0 μg, RNA 100 pmol/L) was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.

| The assay of glucose uptake and lactic acid production
To determine glucose uptake and lactic acid production, after transfection for 24 hours, culture medium was collected and assayed using the Glucose Assay Kit and Lactate Assay Kit (Biovison, Milpitas, California, USA) following the manufacturer's instructions.

| Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
After transfection for 48 hours, total RNA was isolated from cells

| Western blot
After transfection for 24 hours, cells were lyzed in lysis buffer

| Statistical analysis
Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test with SPSS software (version 17.0; Chicago, IL). The data were obtained from at least three independent experiments and presented as the mean ± standard deviation (SD). P < 0.05 was considered to indicate a statistically significant result.

| miR-181b directly targets PIAS3
We used Targetscan, Pictar, and miRanda to search for the putative transcription binding sites of miR-181b and PIAS3 3′UTR, and the predicted sites were shown in Figure 1A. miR-181b mimic or inhibitor was transfected into the cells to overexpress or knockdown miR-181b expression, respectively. As demonstrated in Figure 1B

Warburg effect in colon cancer cells
To reveal the effect of miR-181b on aerobic glycolysis in colon cancer cells, miR-181b inhibitor was transfected into the HCT 116 cells to knockdown miR-181b, and then glucose and lactic acid were detected using the commercial kits. As shown in Figure 2A, knockdown of miR-181b significantly decreased the glucose consumption and lactic acid production. pFlag-PIAS3 plasmid was transfected into the HCT 116 cells to overexpress PIAS3, and the results from western blot demonstrated that compared with the control cells, the relative protein level of PIAS3 was obviously increased in the cells following transfection with the pFlag-PIAS3 plasmid ( Figure 2B).
Further, we found the glucose consumption and lactic acid production were significantly inhibited in the PIAS3-overexpressing cells ( Figure 2C).
To investigate whether PIAS3 mediated the effect of miR-181b on aerobic glycolysis in colon cancer cells, we synthesized three PIAS3 siRs to knockdown PIAS3 and found that PIAS3 siR-3 was the most efficient siRNA ( Figure 2D). Subsequently, PIAS3 siR-3/Scr siRNA were cotransfected with as-miR-181b into the HCT 116 cells, and the glucose consumption and lactic acid production were determined. As shown in Figure 2E, compared with the control, HCT 116 cells with suppressed expression of PIAS3 showed significantly increased glucose consumption and lactic acid production.

| PIAS3 reverses the effect of miR-181b on xenograft tumour growth
To investigate the effect of miR-181b on xenograft tumour growth, HCT 116 cells transfected with as-miR-181b or as-miR NC were injected into the nude mice, and the tumour growth was monitored and measured. It was observed that the tumour volume was significantly decreased in miR-181b-knockdown tumours compared with that of the control tumours (Figure 3A).As shown in Figure 3B, the tumour volumes of xenograft tumours with a high level of PIAS3 expression were decreased compared with that of the control tumours. Furthermore, we found the tumour volume in the as-miR-181b + PIAS3 siR group F I G U R E 3 siPIAS3 Reverses the Effect of miR-181b on Xenograft Tumour Growth. A, miR-181b knockdown inhibits xenograft tumour growth. B, PIAS3 overexpression inhibits xenograft tumour growth. C, siPIAS3 reverses the effect of miR-181b on xenograft tumour growth was larger than that in the as-miR-181b + Scr siR group (Figure 3C).

| PIAS3 reverses the effect of miR-181b on the phosphorylation of STAT3
The miR-181b and miR NC were transfected into the HCT 116 cells.
The as-miR-181b and as-miR NC were transfected into the HT-29 cells. Western blot was performed to examine the protein expression of STAT3 and p-STAT3. As shown in Figure 4A, transfection with the miR-181b significantly increased the relative protein level of p-STAT3. Figure S1 showed that transfection with the as-miR-181b significantly decreased the protein level of p-STAT3. However, the promotive effect of miR-181b on the phosphorylation of STAT3 was reversed by transfection with pFlag-PIAS3 ( Figure 4B).

| PIAS3 overexpression suppresses p-STAT3 and miR-181b expression
The pFlag-PIAS3 was transfected into the HCT 116 and HT-29 cells to overexpress PIAS3. Subsequently, the protein expression of STAT3 and p-STAT3 was examined by western blot, and the miR-181b expression was detected by RT-qPCR. We found PIAS3 overexpression could suppress the p-STAT3 and miR-181b expression ( Figures 5 and S2).

| STAT3 suppression down-regulates miR-181b and up-regulates PIAS3
To knockdown STAT3 expression in HCT 116 cells, three STAT3 siRs were transfected into the cells, and the results of western blot showed that the relative protein level of STAT3 was obviously decreased in STAT3 siR-transfected cells ( Figure 6A). Among these siRs, STAT3-siR3 was the most efficient. Therefore, we selected    Figure 7). We investigated the relationship between miR-181b, PIAS3, and STAT3 expression, and the results from the overexpression and knockdown experiments provided support for our hypothesis. This hypothesis not only clarified the mechanism underlying the role of miR-181b in the activation of STAT3, but also provided a theoretical basis for the persistent activation of STAT3 in colon cancer.
Based on our animal experiment results in vivo, simple intraperitoneal injection of the various constructs can indeed affect tumours derived from cells which were not previously (i.e. in vitro) transiently transfected, but it is necessary to further study whether it can be applied to clinical treatment (or whether intraperitoneal injection is equally effective for clinical treatment). A previous publication revealed that JAK/STAT3 inhibitors could down-regulate the miR-181b expression in oesophageal cancer cells and it was inducible by cytokines activating STAT3. 42 In colon cancer cells, to date, there is no clear answer to whether JAK can affect miR-181b expression and whether it is induced by STAT3 activator, which needs to be further studied. We will explore this issue in next study.