INPP1 up‐regulation by miR‐27a contributes to the growth, migration and invasion of human cervical cancer

Abstract Inositol polyphosphate‐1‐phosphatase (INPP1) is an enzyme that is responsible for glycolysis and lipid metabolism. Here, we discovered that INPP1 expression was up‐regulated in CC tissues compared to that in adjacent normal tissues by RT‐qPCR. Inositol polyphosphate‐1‐phosphatase overexpression promoted and INPP1 knockdown suppressed cell viability, cellular migration/invasion and EMT in CC cells. To explore the mechanism of dysregulation, INPP1 was predicted to be a target of miR‐27a, and a pmiRGLO dual‐luciferase reporter assay showed that miR‐27a bound to the 3′ UTR of INPP1. RT‐qPCR revealed that miR‐27a was also up‐regulated and had a positive correlation with INPP1 expression in CC tissues. Furthermore, shR‐INPP1 could favour the malignant phenotype reversion induced by miR‐27a, suggesting that miR‐27a up‐regulates INPP1 to promote tumorigenic activities. Altogether, our findings show that the up‐regulation of INPP1 by miR‐27a contributes to tumorigenic activities and may provide a potential biomarker for CC.


| INTRODUC TI ON
Cervical cancer (CC) is the third most common leading cause of cancer death among females in developing countries. 1 More than 274 000 individuals die each year. 2 In China, approximately 98 900 new cases were diagnosed in 2015, which accounted for 18.7% of the global incidence. 3 Additionally, 30% of patients were <44 years old. 4 Reportedly, the 5-year survival rates for locally advanced CC vary as follows: 58% for stage IIB, 35% for IIIA, 32% for IIIB and 16% for stage IVA. 5 Therefore, it is urgent to identify novel molecular markers to improve the early diagnosis rate of CC and to develop an effective treatment strategy.
Inositol polyphosphate-1-phosphatase (INPP1) is an enzyme that dephosphorylates free polyphosphorylated inositols, 6 key molecules involved in phosphatidylinositol signalling pathways. 7 Presently, it has been reported that INPP1 is up-regulated in human colorectal cancer. 8 Daniel et al 9 also found that INPP1 is highly expressed in aggressive melanoma, prostate, ovarian and breast cancer cells to promote migration and invasion; this finding suggests that INPP1 may function as an oncogene. However, the role of INPP1 in CC is still unclear.
MicroRNAs (miRNAs) are small non-coding RNAs that can bind to the 3′ UTR of target mRNAs, resulting in translational repression or degradation to regulate a wide range of physiological and pathological processes. [10][11][12] Recent findings demonstrate that miRNA also binds to the 3′ UTR of mRNAs to raise the expression levels of their target genes. [13][14][15] Furthermore, accumulating evidence has indicated that the miRNA profiles were differentially expressed in human cancerous tissues and adjacent normal tissues. 16,17 The dysregulated miRNAs may function as tumour suppressors or oncogenes, 18 which present diagnostic and prognostic values in clinical applications. 19,20 miR-27a was found to be up-regulated and to promote malignancy in non-small-cell lung cancer, breast cancer, prostate cancer, liver cancer, CC, etc. [21][22][23][24][25] In this report, we show that INPP1 is up-regulated in CC tissues compared with the levels in adjacent normal tissues. Inositol polyphosphate-1-phosphatase promoted cell proliferation and invasion/

| Plasmid construction
The miR-27a precursor was cloned from the genome by PCR

| Western blot analysis
The protein samples were obtained by using radioimmunoprecipita-
The details were described previously. 27

| Wound healing and cell invasion assays
For the wound healing assay, the movement of cells into the scraped area was measured to determine cell migration at 0, 48 and 72 hours as described by Ref. 25 For the invasion assays, the cells at the lower surface of the filter were counted as described previously. 28

| Statistical analyses
The significant differences between the sample means obtained from the three independent experiments were analysed using twotailed Student's t tests. Multiple group comparisons were performed by two-way analysis of variance (ANOVA). Pearson's correlation was used to assess the association between two factors. P ≤ .05 was considered statistically significant.

| INPP1 is up-regulated in cervical cancer tissues
To expression and promoted E-cadherin protein expression ( Figure 2F); this finding indicates that INPP1 was able to promote EMT in HeLa and C33A cells. We also obtained similar results in SiHa cells ( Figure   S1). Altogether, these results indicate that INPP1 may function as an oncogene in CC cells.

| miR-27a targets and up-regulates INPP1 expression
To study the potential mechanisms underlying the dysregulation of showed that miR-27a was up-regulated in these CC tissues compared with that in the adjacent normal cervical tissues ( Figure 3A).
The mean expression of miR-27a was increased by approximately 14-fold ( Figure 3B). Furthermore, miR-27a also displayed a positive correlation with INPP1 ( Figure 3C). To alter the miR-27a expression level, the miR-27a expression plasmid pri-miR-27a and the antisense oligonucleotide ASO-miR-27a were used to overexpress or block the expression of miR-27a in CC cells. After the transfection efficiency of pri-miR-27a and ASO-miR-27a was confirmed ( Figure 3D

| Knockdown of INPP1 abolishes the malignant phenotype induced by miR-27a in cervical cancer cells
To verify that INPP1 indeed acts as the functional target of miR-  Figure S2C) and migration ( Figure 4D, Figure S2D). Furthermore, the abnormal expression of EMT markers caused by miR-27a was also restored to normal levels after INPP1 knockdown ( Figure 4E). Taken together, these data suggest that INPP1 is a functional target of miR-27a. The dysregulation of miRNAs has been found to play key critical roles in up-or down-regulating the expression of its target gene 30 and may be a marker of progression in CC. 31 To determine the potential promotes cell viability, colony formation, migration, invasion and EMT in CC cells and implies its potential role as a biomarker in CC.

ACK N OWLED G EM ENTS
This work was supported by the key project of the Committee of Health of Tianjin (No.: 16KJ111).

CO N FLI C T O F I NTE R E S T
None declared.