MicroRNA-155-5p promotes tumor progression and contributes paclitaxel resistance to chemotherapy via TP53INP1 in human Breast Cancer CURRENT

Background MicroRNAs (miRNAs) are a class of small, non-coding RNAs that functioned by targeting specific mRNA. Many studies have reported that, miRNAs play a vital role in cancer progression, they can exert their functions as oncogenes or cancer suppressors. As suggested in previous report, miR-155-5p is up-regulated in breast cancer. Results Here, we found that miR-155-5p promoted cell proliferation and inhibited cell apoptosis in MCF-7. In addition, these effects were reversed when high levels of miR-155-5p were suppressed in MCF7/PR. We determined that TP53INP1 served as a direct target of miR-155-5p through bioinformatic and luciferase assay. These findings confirmed that miR-155-5p was a tumorigenic factor that acted by targeting TP53INP1 and promoted tumor progression and contributed paclitaxel resistance to chemotherapy. Conclusions These results contribute to our understanding of the molecular mechanism underlying breast progression and resistance to

predict higher adverse survival rates and recurrence in several tissue subtypes [9][10][11][12]. Several studies have reported that the overexpression of miR-155-5p in malignant breast tumors is negatively correlated with the overall survival of breast cancer [13,14]. Therefore, further research is needed to better characterize miR-155-5p expression and the function involved in breast cancer progression and chemotherapy resistance. In this study, we aimed to analyze miR155-5p expression profiles in breast cancer and explore the role as well as potential mechanisms of miRNA and target genes in cell migration, invasion and ineffective chemotherapy of breast cancer.

Results
The expression of miR-155-5p is up-regulated in breast cancer To investigate the role of miR-155-5p in the pathogenesis of human breast cancer, we first determined the levels of miR-155-5p expression by in situ hybridization in another series of 57 breast cancer patients, for which clinical and prognosis data were available. This study was discussed and approved by the Ethics Committee of the First Affiliated Hospital of Bengbu Medical College, and all patients signed informed consent. We then analyzed the association between miR-155-5p and the clinic-pathological parameters of these breast cancer patients (Table 1). We found that expression of miR-155-5p was not statistically associated with age, cancer type. However, high miR-155-5p expression was associated with tumor grade, lymph node metastasis,and poor therapy response to paclitaxel. Table 1 The clinic-pathological characteristics of Breast Cancer patients and association with miR-155-5p TP53INP1 was a direct target gene of miR-155-5p TargetScan v7.1, miRanda, PITA, miRmap and miRTarbase were used to predict targets of miR- 155-5p   and 8 Top potential target genes (CEBPB, TP53INP1, MYOD, TAF5L, RNF2, KCTD3, IRF2BP2, EDEM3) were concentrated with lower p-value and FDR (False positive rate) by Venn diagram and detected by qRT-PCR analysis after a transfection with miR-155-5p mimics and inhibitor. And TP53INP1 was identified as a potential target in MCF-7 ( Fig. 2A). Next, qRT-PCR and Western blot were applied in determining the mRNA and protein expression levels of the potential target gene TP53INP1 when transfected with miR-155-5p mimics, inhibitor or negative control. As shown in Fig. 2B, the expression of TP53INP1 was significantly suppressed by miR-155-5p mimics while miR-155-5p inhibitor restored TP53INP1 expression, both at mRNA and protein levels. Then we performed luciferase reporter assay to determine whether TP53INP1 was a direct target of miR-155-5p in breast cancer cells. The target region sequence of the TP53INP1 3'UTR (WT 3'UTR) or a mutant sequence containing putative miR-155-5p sites (MUT 3'UTR) was cloned into a luciferase reporter vector. As expected, the expression of miR-155-5p was strongly enhanced and the dual-luciferase activity was significantly suppressed in cells co-transfected with miR-155-5p and WT-TP53INP1-3'UTR vector, but not the mutated one ( Fig. 2B). To further determine whether TP53INP1 is indeed a direct target of miR-155-5p, we first examined the correlation between miR-155-5p and TP53INP1 mRNA expression in the 528 breast cancer patient dataset from TCGA Data portal. We found statistically significant inverse correlation between miR-155-5p and TP53INP1 expression in these patient samples (Pearson r= -0.42 P < 0.0001;Spearman r= -0.308, P < 0.0001) (Fig. 2C). Therefore, to investigate the specific molecular mechanism of TP53INP1 in regulating breast cancer, expression of TP53INP1 in 1123 breast cancer tissues and 111 normal breast tissues from the TCGA Data portal was analyzed while the mRNA and protein levels of TP53INP1 were analyzed through qRT-PCR and Western blot, respectively. We found that TP53INP1 was significantly down-regulated in breast cancer tissues compared with that in normal breast tissues (P < 0.0001) (Fig. 2D) and that high TP53INP1 expression was correlated with long survival time relative to the low TP53INP1 levels (log rank P < 0.0001). Meanwhile, TP53INP1 was down-regulated in MCF-7/PR compared with MCF-7(P < 0.001) (Fig. 2E).
Ectopic expression miR-155-5p modulateed breast cancer cell apoptosis, migration, and invasion To understand the individual biological function of miR-155-5p in breast cancer, miR-155-5p mimics   were transfected into MCF-7 cells whereas the miR-155-5p inhibitor was transfected into MCF-7/PR   cells. Thereafter the cell apoptosis, cell cycle, migration, and invasion were examined, qRT-PCR and Western blot were carried out to analyze the apoptosis-related genes such as Bcl-2, Caspase-3, Bak-1, Bax and target gene TP53INP1. According to Fig. 3A, miR-155-5p mimics restored the expression of the anti-apoptotic gene Bcl-2 by decreasing the expression of pro-apoptotic genes Bak-1, Bax and

TP53INP1 expression modulateed breast cancer cell growth, migration, and invasion
The strong associations between TP53INP1 and miR-155-5p in breast cancer prompted us to investigate the effect of miR-155-5p-dependent regulation of TP53INP1 on cells apoptosis, migration and invasion abilities. As shown in Fig. S1, qRT-PCR and western blot were conducted to verify the efficiency of specific siRNA and pc-DNA3.1 constructor. Compared with negative control group, knockdown of TP53INP1 promoted cells growth, weakened the ability of migration and invasion, which was associated with an increase in Bcl-2 and a decrease in apoptotic gene levels ( Fig. 4A and 4B). Cell cycle analysis showed that TP53INP1 silencing caused an increase in MCF-7 cells at S phase (from 13.0 to 18.8%) and a reduction of cells at G 1 phase (from 69.3 to 57.1%). Furthermore, apoptosis analysis revealed a lower apoptosis rate (2.05 compared with 7.00%) (Fig. 4C). However, overexpression of TP53INP1 positively promoted apoptosis and inhibited the migration and invasion abilities of MCF-7/PR cells, which was in contrast to the results obtained after over-expressing miR-155-5p ( Fig. 4D-F).
Synergistic effect of combined miR-155-5p and TP53INP1 overexpression therapy Since TP53INP1 has been previously shown to be a targetable marker associated with cell apoptosis, migration and invasion, MCF-7/PR cells co-contransfected with miR-155-5p inhibitor and pc-DNA-TP53INP1 should be an effective method in molecular therapy of cancer. In the present study, we sought to experimentally evaluate whether the dual accumulation of TP53INP1 by miR-155-5p inhibitor and pc-DNA3.1 plasmid showed a synergistic effect on the biological activities of MCF-7/PR cells. As shown in Fig. 5A and 5B, the combination of miR-155-5p inhibitor and pc-DNA-TP53INP1 showed a significant TP53INP1 accumulation, more specifically, similar effects appeared in the oncogenes Bak-1, Bax and Caspase-3, while the expression of Bcl-2 was exactly the opposite, invasion, apoptosis and cell cycle was shown in Fig. 5D. In this study, we have introduced the concept of superimposed effects and synergistic effects. When the effect of combined treatment is significantly higher than the simple superposition of the effects of two independent factors, these effects should be considered as a synergistic effect. Further analysis found that our combination therapy has obvious synergistic effects in inhibiting cells invasion and promoting apoptosis based on Chou-Talalay combination index equation. As for cell migration and cell cycle distribution, it only showed a superimposed effect.
Therefore, our evidence indicates that miR-155-5p has important roles in cancer cell resistance to chemotherapy drugs.

Discussion
Recent studies have confirmed that miRNAs play a fatal role in the progression of several cancers, which also serve as the molecular markers for tumor diagnosis, treatment, and efficacy [15][16][17][18][19]. It has been many years since the earliest miRNA lin-4 and let-7 have been investigated, and this seems to be a far-reaching study although the mechanism of action of miRNAs has been described due to the complex and variable genetic regulatory network. In this research, we used a large-scale cancer genome database and bioinformatics to screen and validate the dysregulation of miR-155-5p in breast cancer. Then it was confirmed that TP53INP1 was a direct target of miR-155-5p. We explored the ability of miR-155-5p in modulating cells apoptosis, migration and invasion by repressing the target gene TP53INP1 in MCF-7 and MCF-7/PR cells. In our study, we found that miR-155-5p expression was significantly up-regulated in breast cancer tissue compared with normal breast tissues. Moreover, we discovered that upregulation of miR-155-5p significantly stimulated cell growth and enhanced cell proliferation and migration, and promotes resistance of paclitaxel therapy which contributes to ineffective drug chemotherapy. However, it remains unclear about the specific mechanism by which miR-155-5p regulates the biological activity of breast cancer cells. Tumor protein 53-induced nucleoprotein 1 (TP53INP1) is a target gene of p53 that is induced by pro-apoptotic stress. It has been reported that TP53INP1 plays an important role in p53-mediated apoptosis and cell cycle arrest through the p53-dependent and p53-independent pathways [20,21]. In addition, relevant reports point out that the expression of TP53INP1 is significantly down-regulated or lost during the development of several cancers such as liver cancer, NSCLC, and pancreatic cancer [22][23][24]. Wang et al. reported that TP53INP1, as a target gene for both miR-19a and miR-19b, TP53INP1 induced tumor cell death as a key tumor suppressor for p53, and p53 directly bound to the TP53INP1 promoter and triggered TP53INP1 in vivo accumulation [25]. In addition, it is reported that the positive rate of TP53INP1 decreases as gastric cancer progresses, and the pathological phenotype of invasive gastric carcinoma is significantly correlated with the low positive rate of TP53INP1 [26,27]. The discovery of miRNAs has revolutionized research in gene expression and functional genomics. As we all know, altered metabolism of anticancer drugs is correlated with drug resistance. In clinical situations, acquired drug resistance frequently follows chemotherapeutic regimens and is considered the major cause of mortality in Breast Cancer [31][32][33][34][35]. Although paclitaxel has been considered the first-line chemotherapy drug for breast cancer, drug resistance leading to chemotherapy failure is almost unavoidable [36]. Increasing studies have suggested that miRNA is a new class of therapy molecule, and has a more modulatory role than traditional drugs.

Conclusions
It can be concluded that miR-155-5p can be used as a good biomarker for diagnosis and monitoring of prognosis. The combination of miR-155-5p and TP53INP1 is an effective way to strongly inhibit tumor growth and target organ metastasis. At the same time, the sensitivity of breast tumors to the chemotherapy drug paclitaxel was restored.

Migration assay
Cell migration was determined through the wound healing assay, which was also called the "scratch" assay. In brief, cells were digested with 0.25% trypsin, centrifuged, and prepared into the single cell suspension. Cell suspension was cultured into 6-well plate until the cell density reached 80%-90%. A wound track was introduced by scraping the cell monolayer with the germ-free pipette tip(10 µl).
Attention should be paid to keep the same width of these cuts, then, cells were washed twice with PBS, and treated with related dose. The average extent of wound closure was quantified in 3 random fields with × 50 magnification at 0 and 24 h, respectively. All microscope images were obtained with the Olympus IX73 (Olympus Corporation, Japan).

Invasion assay
For the invasion assay, cells were digested with 0.25% trypsin, prepared into single cell suspension with serum-free medium and the cell density of each group was maintained same. According to the experimental requirements, each group of cells ( In order to test the cell's resistance to paclitaxel, we tested the cell viability at different concentrations, using these data to plot XY and fit the data with a straight line (linear regression).
IC50 value was then estimated using the fitted line. All these analyses and the production of pictures are done in Graphpad Prism.

Statistical analysis
All experiments were performed for at least three times, and datas were statistically analyzed using the unpaired, two-tailed Student's t-tests followed by Newman-Keuls test built into GraphPad Prism (GraphPad Software 8.0.1; San Diego, CA). All data were expressed as mean ± SE. Furthermore, all statistical differences included a 95% confidence interval (CI). miR-155-5p promotes paclitaxel resistance by suppressing TP53INP1. a MCF-7 and paclitaxel-resistant cells MCF-7/PR were exposed to different concentrations of paclitaxel for 24 h, and then cell viability was assayed after treatment with various concentrations of paclitaxel for 48 h by SRB assay. b-f paclitaxel-resistant cells MCF-7/PR were co-transfected with miR-155-5p mimic/inhibitor or si-TP53INP1/ pc-TP53INP1 for 24 h, and then cell viability was assayed after treatment with various concentrations of paclitaxel for 48 h by SRB assay.
Each experiment was repeated three times.