A Novel Mirna Identified in GRSF1 Complex Drives the Metastasis Via the PIK3R3/AKT/NF-Κb and TIMP3/MMP9 Pathways in Cervical Ca

Sun et al. Cell Death and Disease (2019) 10:636 https://doi.org/10.1038/s41419-019-1841-5 Cell Death & Disease

ARTICLE Open Access AnovelmiRNAidentiﬁed in GRSF1 complex drives the metastasis via the PIK3R3/AKT/NF-κBand TIMP3/MMP9 pathways in cervical cancer cells Qi Sun1, Zhen Yang1,PuLi2,XuWang1,LuSun1, Shixing Wang1,MinLiu1 and Hua Tang1

Abstract microRNAs (miRNAs) play an important role in carcinogenesis. Typically, miRNAs downregulate the target expression by binding to the 3′ UTR of mRNAs. However, recent studies have demonstrated that miRNAs can upregulate target gene expression, but its mechanism is not fully understood. We previously found that G-rich RNA sequence binding protein (GRSF1) mediates upregulation of miR-346 on hTERT gene. To explore whether GRSF1 mediate other miRNA’s upregulation on their target genes, we obtained profile of GRSF1-bound miRNAs by Flag-GRSF1-RIP-deep sequencing and found 12 novel miRNAs, named miR-G. In this study, we focused on miR-G-10, which is highly expressed in cervical cancer tissues and cell lines and serum from patients with metastatic cervical cancer. miR-G-10 in cervical cancer cells significantly promoted migration/invasion and anoikis resistance in vitro and lung metastasis in vivo. Furthermore, miR-G-10 bound to the 3′ UTR of PIK3R3 and upregulated its expression to activate the AKT/NF-κB signal pathway in a GRSF1-dependent manner, whereas miR-G-10 suppressed TIMP3 in the AGO2 complex to modulate the MMP9 signaling pathway in cervical cancer cells. Taken together, our findings may provide a new insight into the upregulation mechanism mediated by miRNAs and a potential biomarker for cervical cancer. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,;

Introduction been investigated7, the explicit developmental process of Cervical cancer (CC) is the fourth most common cervical carcinogenesis is still mostly unclear. malignancy and a crucial cause of cancer-associated Emerging evidence has revealed that microRNAs (miR- mortality in women1,2. Up to now, the standard strate- NAs) are involved in cervical cancer development, pro- – gies for patients with cervical cancer are surgery resection, gression, clinical outcome, and treatment response8 10. chemotherapy, and radiotherapy3. Enhanced pathological Given that most miRNAs suppress targets expression by examination of cervical cancer tissue and improved binding to the 3′ UTRs of mRNAs; however, some inter- inchoate diagnosis have signiﬁcantly reduced the mor- esting studies have demonstrated that miRNAs can upre- bidity and mortality of this disease in the past decades4,5. gulate target gene expression at the posttranscriptional However, there are ~528,000 new cases and 266,000 level11,12. For example, miR-369-3p promotes TNFα deaths every year in the world6. Although the certain translation through AU-rich elements (AREs)13,miR-490- molecular mechanisms of cervical carcinogenesis have 3p upregulates endoplasmic reticulum–Golgi intermediate compartment 3 (ERGIC3) in human hepatocellular carcinoma cells14. Recently, we elucidated that G-rich RNA sequence binding protein (GRSF1) mediates miR-346 to Correspondence: Hua Tang ([email protected]) 1Tianjin Life Science Research Center and Department of Pathogen Biology, enhance expression of human telomerase reverse tran- Collaborative Innovation Center of Tianjin for Medical Epigenetics, School of scriptase (hTERT) in cervical cancer cells15.miR-23apro- Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China α 2 motes IKK expression to contribute to the malignancy of Tianjin Central Hospital of Gynecology and Obstetrics, Tianjin 300100, China 16 These authors contributed equally: Qi Sun, Zhen Yang, Pu Li epithelial ovarian cancer cells .However,themolecular Edited by G. Calin

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mechanism underlying miRNAs upregulating the expres- detect its’ level in various cancer cell lines. As shown in sion of the target genes is not completely understood. Fig. 1f, all these cell lines expresses miR-G-10, but it was GRSF1 is a family member of heterogeneous nuclear highest level in cervical cancer cell lines and lowest level ribonucleoprotein (hnRNP) F/H, which specifically binds in the immortalized cervical epithelial S12 cells. In addi- to G-rich RNA sequences (G-tracts) to take part in spli- tion, we examined miR-G-10 expression in various cer- cing regulation of mRNAs17. In addition, GRSF1 regulates vical cancer cell lines. The expression level of miR-G-10 translation of viral RNA and eukaryotic transcripts in the in HeLa and C33A cells is relatively higher than that in – cytoplasm18 20. In the mitochondria, GRSF1 has been the other cervical cancer cells expect SiHa (Fig. 1g). Then, detected in dynamic mitochondrial granules (MRGs)21, we detected the level of miR-G-10 in 21 paired cervical and regulates differentiation of mammalian embryonic cancer tissues and the adjacent nontumorous tissues by stem cells22. More recently, Guo et al. and Noh et al. have RT-qPCR. The clinical information of patients are in reported that GRSF1 is implicated in processing of reg- Supplementary Table 1. Similarly, miR-G-10 level was ulatory RNAs23,24, and may be involved in tumorigenesis upregulated in 17 paired cervical cancer tissues (Fig. 1h). in cervical cancer. In addition, we found that the precursor of miR-G-10 To determine whether GRSF1 is involved in regulation located in the Chromosome 5 (136129333, 136129440), of other miRNAs which contributes oncogenic activity, which is the noncoding region of human SMAD family we previously found that 630 miRNAs are associated with member 5 (SMAD5). Accordingly, we examined the GRSF1 in cervical cancer cells by GRSF1-RIP-deep mRNA level of SMAD5 in cervical cancer tissues and the sequence. Here, we identified a novel GRSF1-bond adjacent nontumorous tissues, and S12 cells, HeLa cells miRNA, miR-G-10 (GenBank accession number: Homo by RT-qPCR. As shown in Fig. 1i, j, the expression level of MN153037), and determined its oncogenic role in cervical SMAD5 was lower in cancer tissue and cells than normal. cancer cells. Furthermore, we demonstrated that phos- The analysis of TCGA database supported the same phatidylinositol 3-kinase regulatory subunit gamma results (Fig. 1k). Which means that, the high level of miR- (PIK3R3) and tissue inhibitors of metalloproteinase 3 G-10 in cervical cancer was not caused by its host gene. (TIMP3) are direct targets of miR-G-10 and identify them Overall, these results indicate that miR-G-10 is upregu- as crucial regulators of the AKT/NFκB and MMP9 lated in cervical cancer cells. pathways, respectively. Finally, we proved that miR-G-10 upregulates PIK3R3 in a GRSF1-dependent manner, but miR-G-10 promotes oncogenic activity in vitro and vivo downregulates TIMP3 in an AGO2 pathway to promote To explore the role of miR-G-10 in cervical cancer cells, malignancy of cervical cancer cells. we generated the overexpression and knockdown plas- Collectively, our findings have shed light on mechanism mids of miR-G-10. The efficiency of the indicated plas- by which miRNA modulates targets function in tumor- mids measured by RT-qPCR in HeLa and C33A cells igenesis and may have a potential value for clinical (Fig. 2a). To determine the effect of miR-G-10 on cell application in patients with cervical cancer. proliferation, HeLa and C33A were transiently transfected with miR-G-10, anti-miR-G-10, or controls. MTT assay Results (Fig. 2b), EdU incorporation assay (Fig. 2c), and colony- Profiling GRSF1-bond miRNAs and validation of miR-G-10 formation assay (Fig. 2d) showed that miR-G-10 did not Our previous study delineated that GRSF1 enhances significantly affect the viability and proliferation of HeLa hTERT expression via binding its 3′ UTR. To determine and C33A cells. To further explore whether miR-G-10 whether GRSF1 binds to miRNAs which upregulate tar- could mediate tumor growth, we performed transplanta- get’s gene expression, we utilized RNA-deep sequencing tion tumor experiment in nude mice. As shown in Fig. 2e, to profile miRNAs isolated from Flag-GRSF1 to identify miR-G-10 can promote the growth of tumor in vivo. the GRSF1-bond miRNAs and their functions in cervical Interestingly, the results of transwell and wound-healing cancer cells. As shown in Fig. 1a, total 630 miRNAs were assays indicated that the migration and invasion ability found in the complex, 618 known miRNAs, and 12 novel were significantly enhanced by miR-G-10 overexpression, miRNAs, which we named GRSF1-bond miRNAs (miR- and restrained by anti-miR-G-10 transfection in vitro G). The total read counts of deep sequence showed that (Fig. 2f, g). To investigate the role of systemic miR-G-10 miR-G-10 is the most abundant with 617 reads among the intervention in vivo, a xenograft model in nude mice was novel miRNAs (Fig. 1b, c). Thus, we chose miR-G-10 for established. It was observed that mice injected with miR- further investigation. First, we predicted the secondary G-10 cells had a higher number of tumor nodules, and structure of the classic hairpin stem–loop structure of formed larger tumors when compared with the control miR-G-10 precursor (Fig. 1d). Northern blot assay con- (Fig. 2h). H&E staining showed that the lung tissues in the firmed the precursor and mature of miR-G-10 in HeLa miR-G-10 group have typical characteristics of tumor, and C33A cells (Fig. 1e). Then, we applied RT-qPCR to that is, the nuclei are large, deeply stained, and cells are

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Fig. 1 Profile of GRSF1-bond miRNAs and validation of miR-G-10. a The percentage of new miRNA and known miRNA in deep sequence results. b The read count of novel miRNAs. c The mature miRNA sequence and deep sequence data of miR-G-10. d Secondary structure prediction of miR-G- 10. e Northern blot was performed using the biotin-conjugated antisense strand of miR-G-10 as a probe. The graph of PAGE gels is on the left, the top is xylene cyanol, and the bottom is bromophenol blue, representing 50 nt and 15 nt, respectively. Lane 1: 0.1pM miR-G-10-biotin probe. Lane 2: 30 μg of small RNA from HeLa cell; lane 3: 30 μg of small RNA from C33A cells. f miR-G-10 expression in different kinds of cancer cell lines. g miR-G-10 expression in various cervical cancer cell lines. h miR-G-10 expression in human cervical cancer tissues and paired adjacent tissues. i SMAD5 expression in human cervical cancer tissues and paired adjacent tissues. j SMAD5 expression in S12 cells and HeLa cells. k The expression analysis of SMAD5 in TCGA database. The expression levels were normalized to U6 snRNA. All of the experiments were repeated three times. *P < 0.05; **P < 0.01; ***P < 0.001. NS not significant closely arranged, while the control group had less expression level of miR-G-10 in the serum samples of tumorous tissues (Fig. 2i). miR-G-10 embodied sig- cervical cancer patients by RT-qPCR, and found that its nificantly advantages to promote tumor metastasis com- expression in the patients with metastatic nodules were pared with control group in vivo. Next, we examined the higher than that of patients without metastasis (Fig. 2j).

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Fig. 2 (See legend on next page.)

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(see ﬁgure on previous page) Fig. 2 miR-G-10 promotes oncogenic activity in cervical cancer cells. a Relative level of miR-G-10 after transfection with miR-G-10 or Anti-miR-G- 10 in HeLa and C33A. b MTT assay, c EdU assay (scale bar: 20 μm), and d colony-formation assay (scale bar: 5 mm) were used to detected cell proliferation. e Representative graph of tumors (left) and tumor weights (right). f Transwell assays showed the invasion tendency. Scale bar: 50 μm. g Wound-healing assays were used to measure migration. Scale bar: 50 μm. h In vivo metastasis assay. Representative lungs were harvest after injecting HeLa cells with pcDNA3 or miR-G-10 overexpression. i Representative H&E staining results of metastatic nodules in the lungs are shown. Scale bar: 50 μm. j miR-G-10 levels in serum from cervical cancer patients with/without metastasis. The expression levels were normalized to U6 snRNA. All of the experiments were repeated three times. *P < 0.05; **P < 0.01; ***P < 0.001. NS not signiﬁcant

Taken together, these results suggested that miR-G-10 TIMP3 inhibits the migration and invasion in cervical promoted the migration/invasion and metastasis of cer- cancer cells vical cancer in vitro and in vivo. Emerging evidences demonstrated that TIMP3 plays a – crucial role in several cancers25 28. It has been confirmed miR-G-10 upregulates PIK3R3, but downregulates TIMP3 that TIMP3 plays an inhibitor role in the proliferation of expression cervical cancer cells29. However, the other function of To uncover the molecular mechanisms by which miR- TIMP3 in cervical cancer remains unclear. To further G-10 exerts oncogenic effects on cervical cancer, can- investigate the effects of TIMP3 on cell malignant beha- didate targets of miR-G-10 were predicted by TargetS- viors, we first constructed TIMP3 overexpression vector can and miRDB. According to the bioinformatics assay (pcDNA3/TIMP3) and knockdown plasmid (shR- and the functional knowledge of the candidates, we TIMP3). The efficiency was also confirmed by RT-qPCR chose PIK3R3 and TIMP3 as targets for further study. and western blot analysis (Fig. 4a, b). The results of First, bioinformatics analysis predicted that the 3′ UTR transwell and wound-healing assays showed that TIMP3 of PIK3R3 and TIMP3 transcripts contain the com- inhibited the ability of migration and invasion in HeLa plementary site of miR-G-10. To confirm that miR-G-10 and C33A cells (Fig. 4c, e). We also detected an EMT directly targets PIK3R3 and TIMP3, EGFP reporter marker by western blot, TIMP3 markedly enhanced the assays were performed, which contain either the wild- expression of epithelial marker (E-cadherin and cytoker- type 3′ UTR or mutational 3′ UTR in the complementary atin), inhibited the expression of mesenchymal marker seed sequences of PIK3R3 and TIMP3 (Fig. 3a, b). miR- (vimentin) (Fig. 4d). To further determine whether the G-10 significantly advanced the EGFP intensity of function of miR-G-10 on aggressive phenotype of cervical PIK3R3-3′ UTR, but repressed the intensity of TIMP3-3′ cancer cells is mediated by regulating TIMP3, we carried UTR (Fig. 3c, d). Nevertheless, mutation in the 3′ UTR out a series of functional rescue experiments. TIMP3 and of the miR-G-10 target sequence led to a complete miR-G-10 co-transfection rescued the inhibition of abrogation of reporter inhibition or promotion (Fig. 3e, TIMP3 caused by miR-G-10 in HeLa cells (Fig. 4f). As f). Furthermore, we found that miR-G-10 overexpression shown in Fig. 4g, overexpression of TIMP3 partly rescued significantly reduced the expression levels of TIMP3, but the malignant behavior of HeLa and C33A cells. These enhanced the PIK3R3 levels at both mRNA and protein results proved that miR-G-10 promotion of migration/ levels (Fig. 3g-j), while anti-miR-G-10 transfection have invasion and EMT were partly by downregulating TIMP3 the opposite role. As known, miRNAs downregulate the in cervical cancer cells. target expression by binding to the 3′ UTR of mRNAs In addition, previous reports demonstrated that multi- – within the RNA-induced silencing complex (RISC). To ple ADAMs and MMPs are regulated by TIMP330 32.To determine whether downregulation of miR-G-10 on determine the regulation mechanism of migration and TIMP3 is dependent on AGO2, we performed an AGO2 invasion are mediated by TIMP3, we examined the level RNA immunoprecipitation (RIP) and differential of ADAM17 and MMP9 in cervical cancer cells. As expression analysis by RT-qPCR assay. As shown in Fig. expected, the expression levels of ADAM17 and MMP9 3k, l, miR-G-10 and TIMP3 mRNA were enriched in the are increased after transfection with miR-G-10, but AGO2-IP fraction in HeLa cell, indicating that miR-G- inhibited by anti-miR-G-10 transfection in HeLa cells 10 suppresses TIMP3 in the AGO2 complex. Accord- (Fig. 4h). Meanwhile, we found that β-integrin, an ingly, we analyzed correlation between miR-G-10 and important adhesion molecule, was also inhibited by miR- TIMP3/PIK3R3. We found that miR-G-10 was nega- G-10 overexpression. Accordingly, pcDNA3/TIMP3 tively correlated with TIMP3, but positively with PIK3R3 resulted in a significantly reduction of ADAM17 and (Fig. 3m, n). Taken together, these data indicate that MMP9. However, cells treated with miR-G-10 and shR- TIMP3 and PIK3R3 are direct targets of miR-G-10 in TIMP3 restored the expression of ADAM17 and MMP9 cervical cancer cells. (Fig. 4i). Thus, these results indicated that TIMP3

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Fig. 3 miR-G-10 upregulates PIK3R3, but downregulates TIMP3 expression. a, b The predicted miR-G-10-binding sites in the 3′ UTR of PIK3R3 and TIMP3 mRNA using miRDB and Targetscan. c HeLa and C33A cells were co-transfected with pcDNA3/EGFP-TIMP3-3′ UTR (wild-type) or (d) pcDNA3/EGFP-PIK3R3 and miR-G-10 or Anti-miR-G-10. e HeLa and C33A cells were co-transfected with pcDNA3/EGFP-TIMP3-3′ UTR-mut or (f) pcDNA3/EGFP-PIK3R3-3′ UTR-mut and miR-G-10 or anti-miR-G-10. g, h RT-qPCR showed TIMP3 and PIK3R3 mRNA levels after transfection with miR-G- 10 or anti-miR-G-10. i, j Western blot assay showed TIMP3 and PIK3R3 protein levels after transfection with miR-G-10 or anti-miR-G-10. k, l RT-qPCR showed the RNA levels of miR-G-10 and TIMP3 in the AGO2-RIP complex. m, n Correlation analysis between miR-G-10 and TIMP3/PIK3R3. All of the experiments were repeated three times. *P < 0.05; **P < 0.01; ***P < 0.001. NS not signiﬁcant

inhibited cells metastasis through the regulation of the the repressive effect of miR-G-10 on cell invasion and extracellular matrix protein of ADAM17 and MMP9. metastasis in cervical cancer, we performed the following experiments. RT-qPCR and western blot assays validated GRSF1-mediated miR-G-10 upregulates the PIK3R3, and the efﬁciency of overexpression and knockdown plasmids promotes the malignant phenotype of cervical cancer cells of PIK3R3 (Fig. 5a, b). Overexpression of PIK3R3 mark- It has been identiﬁed that PIK3R3 can facilitate tumor edly promotes cell migration and invasion (Fig. 5c). growth of cervical cancer, but its’ effect on metastasis is PIK3R3 overexpression inhibited E-cadherin and cyto- still unclear33. To validate whether PIK3R3 accounts for keratin expression but promoted vimentin expression,

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Fig. 4 (See legend on next page.)

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(see figure on previous page) Fig. 4 TIMP3 inhibits the migration and invasion in cervical cancer cells. a, b RT-qPCR and western blotting showed TIMP3 levels in HeLa C33A cells with overexpress or knockdown plasmid. c Transwell assays showed that TIMP3 suppresses cells’ migration and invasion. Scale bar: 50 μm. d Western blotting showed the protein levels of E-cadherin, vimentin, and cytokeratin after transfection with TIMP3 overexpression or shR-TIMP3. e Wound-healing assays showed that migration was suppressed by TIMP3 overexpression and promoted by shR-TIMP3. Scale bar: 50 μm. f Co- transfection with miR-G-10 and TIMP3 partly rescued the suppression of miR-G-10 on TIMP3 and (g) cell migration and invasion in HeLa and C33A. Scale bar: 50 μm. h The protein levels of MMP9, ADM17, and β-integrin in HeLa and C33A cells transfected with miR-G-10 or anti-miR-G-10. i HeLa cells were transfected with the indicated combinations of pcDNA3, miR-G-10, pcDNA3/TIMP3, and shR-TIMP3. Western blot assay was used to measure the protein level of MMP9 and ADAM17. All of the experiments were repeated three times. *P < 0.05; **P < 0.01; ***P < 0.001. NS not significant indicating that PIK3R3 accelerated EMT process (Fig. 5d). cells in vitro, but it significantly promoted migration/ To further demonstrate the effects of miR-G-10 and invasion and metastasis in vitro and vivo. Due to anoikis PIK3R3 on aggressive phenotype of cervical cancer cells, resistance playing a crucial role in the cancer metastasis, we carried out a series of functional rescue experiments. we investigated regulation of miR-G-10 on anoikis in shR-PIK3R3 rescued the oncogenic role caused by miR- cervical cancer cells. The results of cell-material adhesion G-10 in HeLa and C33A cells (Fig. 5e, f). These results assay showed that miR-G-10 restrained cell–matrix proved that the promotion of miR-G-10 on invasion and adhesion by ~60% (Fig. 6a, left panel), while anti-miR-G- metastasis was partly by upregulating PIK3R3 in cervical 10 enhanced cell–matrix adhesion by ~1.5–2.0-fold cancer cells. Previously, we have revealed that miR-346 (Fig. 6a, right panel). We also found that miR-G-10 promotes the expression of human hTERT in a GRSF1- decreases E-cadherin and cytokeratin but increases dependent manner. To determine whether GRSF1 med- vimentin expression in attached cells (HeLa-att) than iates the upregulation of PIK3R3 expression by miR-G-10, suspension HeLa cells (HeLa-sus) (Fig. 6b, c). In addition, GRSF1-knockdown HeLa cell lines were established. We miR-G-10 greatly decreased the number of apoptotic cells picked eight clones randomly and examined the expres- detected by flow cytometry following cell detachment, but sion of GRSF1 by western blot. Among them, 3#-shR- anti-miR-G-10 was utterly counterproductive (Fig. 6d). GRSF1 cell was the lowest expression of GRSF1 (Fig. 5g). Furthermore, the levels of pro-apoptosis protein cleaved- Gain-of-function and loss-of-function assays were per- caspase 3 and cleaved PARP were markedly decreased, formed in GRSF1-depleted cells transfected with either but the expression levels of Bcl-2 were increased after miR-G-10 or anti-miR-G-10. As shown in Fig. 5h, alter- miR-G-10 transfection in HeLa cells with suspension nation of miR-G-10 has no effect on the protein levels of growth manner (Fig. 6e). Apoptosis analysis showed a PIK3R3 in GRSF1-depleted HeLa cells. Furthermore, we lower apoptosis rate among HeLa cells with suspension performed the rescue experiment. Co-transfection with growth manner when PIK3R3 was overexpressed (Fig. 6f). pcDNA3 and miR-G-10, pcDNA3 and GRSF1, miR-G-10 This reduction was confirmed by western blot-based and GRSF1, which significantly promotes PIK3R3 quantifications of apoptosis-associated gene expression in expression in HeLa cells, but co-transfection with miR-G- HeLa-sus cells. The expression levels of Bcl-2 was sig- 10 and shR-GRSF1 obviously abrogated the upregulation nificantly increased after treated with PIK3R3 in HeLa-sus of miR-G-10 on PIK3R3 level (Fig. 5i). Meanwhile, co- cells, but the expression levels of cleaved caspase 3 and transfection with anti-miR-G-10 and shR-GRSF1 in HeLa cleaved PARP were dramatically reduced (Fig. 6g). cells dramatically reduced PIK3R3 compared with con- Together, our results suggested that miR-G-10 protected trols (Fig. 5i). Moreover, we examined the PIK3R3 mRNA cervical cancer cells from anokis, and may contribute the enriched in the GRSF1 complex by the co- metastasis of cervical cancer cells in vitro. immunoprecipitated RNA (GRSF1-RIP) and RT-qPCR. As shown in Fig. 5j, PIK3R3 mRNA was enriched in the miR-G-10 regulates NF-κB and extracellular matrix GRSF1 complex when compared with the control group. pathways in cervical cancer cells – Taken together, these data indicate that PIK3R3 pro- NF-κB is a downstream of the PI3K/AKT pathway34 36, moted migration/invasion and metastasis, and upregula- which plays a critical role in tumorigenesis. Our above tion of PIK3R3 by miR-G-10 was mediated by GRSF1. results have demonstrated that miR-G-10 targets the 3′ UTR PIK3R3 and enhances its expression. The activation miR-G-10 protects cervical cancer cells from anoikis (p-AKT level) of the PI3K pathway was evidenced by the The resistance of anoikis is manifesting as a trait of increase or decrease level of PIK3R3 by overexpression or cancer cells and contributes to the formation of metastasis knockdown (Fig. 7a). Next, we examined p-AKT levels in in distant organs. Although we did not observe that miR- HeLa cells treated with miR-G-10. Overexpression of G-10 has obvious impact on the viability of cervical cancer miR-G-10 significantly increased, whereas depletion of

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Fig. 5 (See legend on next page.)

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(see figure on previous page) Fig. 5 GRSF1-mediated miR-G-10 upregulates the PIK3R3 and promotes the malignant phenotype of cervical cancer cells. a, b RT-qPCR and western blotting showed PIK3R3 levels in HeLa C33A cells with overexpress or knockdown plasmid. c Transwell assays showed that PIK3R3 promotes cells’ migration and invasion. Scale bar: 50 μm. d Western blotting showed the protein levels of E-cadherin, vimentin, and cytokeratin after transfection with PIK3R3 or shR-PIK3R3. e Co-transfection with miR-G-10 and PIK3R3 showed that PIK3R3 partly rescued the upregulation of miR-G-10 on PIK3R3 and f cell migration and invasion in HeLa and C33A. Scale bar: 50 μm. g Western blot assay was used to determine the protein level of GRSF1-knockdown cells (#1-#8) and HeLa cell as a control. h We examined the protein level of PIK3R3 in GRSF1-knockdown cells (#3) by western blot after transfection miR-G-10 and anti-miR-G-10. i Western blot assay showed the expression levels of PIK3R3 transfected with the indicated plasmids in GRSF1-knockdown cells. j RT-qPCR showed the RNA level of PIK3R3 in the GRSF1-RIP complex. All of the experiments were repeated three times. *P < 0.05; **P < 0.01; ***P < 0.001. NS not significant miR-G-10 decreased the p-AKT level (Fig. 7b). To address dependent manner45,46. However, we previously deli- that miR-G-10 is involved in the activation of the NF-κB neated that miRNA-346 binds to hTERT 3′ UTR and signaling pathway, we first examined the nuclear trans- augments hTERT expression in a GRSF1-dependent location of NF-κB1 by western blot and the results proved manner, which promotes oncologic activity in cervical that miR-G-10 accelerated the process of nuclear trans- cancer cells15. To explore whether other miRNA enhan- location through the PI3K/AKT pathway (Fig. 7c). Over- cer target gene expression through GRSF1, we first expression of miR-G-10 increased the nuclear distribution applied RIP and deep sequencing and found that 618 of NF-κB1, and knockdown of PIK3R3 partly counter- known miRNAs and novel miRNA in the GRSF1 complex acted this promoting effects of miR-G-10 (Fig. 7d). of HeLa cells. Simultaneous, we examined the subcellular location of In this study, we focused on a highest level novel NF-κB1 by immunofluorescence in HeLa cells. Nucleus miRNA, miR-G-10. The criteria for identifying novel accumulation of NF-κB1 was obviously enhanced after miRNA were northern blot, RT-qPCR and bioinformatics miR-G-10 transfection, and inhibited by anti-miR-G-10 analysis for predicating the secondary structure47. First, transfection (Fig. 7e). Besides, the distribution of NF-κB1 we predict the precursor structure of miR-G-10 by in the nucleus was increased when co-transfection with bioinformatics analysis. Then, we used northern blot to pcDNA3 and PIK3R3 in HeLa cells, but this effect was confirm the existence of miR-G-10 and its precursor in declined after co-transfection with miR-G-10 and shR- HeLa and C33A cells. We found that miR-G-10 is PIK3R3 (Fig. 7f). Taken together, our results indicated expressed at high levels in cervical cancer tissues. In that miR-G-10 activated the PI3K/AKT/NF-κB signaling addition, the levels of miR-G-10 are correlated with pathway during the regulation of cervical cancer metastasis in patients with cervical cancer. Next, we used progression. gain-of-function and loss-of-function approaches to reveal that upregulated miR-G-10 facilitates the process of Discussion migration and invasion, and significantly induces the lung Over the past few decades, emerging studies have elu- metastasis of cervical cancer in vivo, but do not have cidated that miRNAs are involved in tumorigenesis and obvious effects on cell proliferation in vitro. Furthermore, – may be targets for novel therapeutic approaches37 39. we evidenced that miR-G-10 promotes the anoikis resis- During the development and progression of cancer, tance of cervical cancer cells. Moreover, miR-G-10 inhi- multiple miRNAs could take part in tumorigenesis and bits cell adhesion in situ and promotes the viability of cells the regulation of tumor microenvironment40,41. For under a state of suspension, which may contribute the example, Hu et al. reported that miR-215 was induced by formation of tumor metastases. the hypoxia, which is vital for glioma-initiating cells to fit As known, most studies on miRNA pathways have been the hypoxic microenvironment via suppressing the focused on gene silencing mechanisms. But recently it has expression of KDM1B and modulating activities of mul- become interesting that some miRNAs also could also tiple pathways42. miR-424(322)/503 as a tumor suppressor active target genes expression via different pathways. For in breast cancer and promotes chemoresistance through example, miR-122 can enhance hepatitis C viral (HCV) upregulate Bcl-2 and insulin-like growth factor-1 receptor gene replication by targeting 5′-noncoding elements in (IGF1R)43. miR-31 promotes mammary stem cell expan- the HCV genome48 and miR-346 binds to 3′ UTR of sion and breast tumorigenesis by suppressing Dkk1 and hTERT transcript and activates it’s expression in a activates Wnt/β-catenin signaling44. It has been known GRSF1-dependent manner15. Bioinformatics analysis that miRNAs regulate the expression of following mole- suggested hundreds of candidate targets for miR-G-10, we cules and make further influence on various significant chose TIMP3 and PIK3R3 as the putative targets for pathways. Typically, miRNA suppresses target gene further study after considering known functional knowl- expression via targeting its 3′ UTR in an AGO2- edge. We identified that miR-G-10 binds to the 3′ UTR of

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Fig. 6 (See legend on next page.)

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(see ﬁgure on previous page) Fig. 6 miR-G-10 protects cervical cancer cells from cell detachment-induced anoikis. a Cell–matrix adhesion assay of HeLa cells with overexpress (left panel) or knockdown (right panel) miR-G-10. Scale bar: 50 μm. b, c Western blot results showed E-cadherin, vimentin, and cytokeratin expression in HeLa cells with attached (att) and suspended (sus) growth manner. d, f FACS analysis of the apoptosis of suspended (sus) HeLa cells after transfection with miR-G-10, anti-miR-G-10, and pcDNA3/PIK3R3, shR-PIK3R3, respectively. e, g Bcl-2, cleaved caspase 3, and cleaved PARP expression level in the indicated plasmids transduced HeLa cells with suspended growth manner. All of the experiments were repeated three times. *P < 0.05; **P < 0.01; ***P < 0.001. NS not signiﬁcant

TIMP3 and PIK3R3 transcripts, and miR-G-10 down- of the Declaration of Helsinki. The specimens were snap- regulates TIMP3 in classic AGO2-dependent manner, frozen immediately after collection, and were stored at whereas upregulates PIK3R3 in a GRSF1-dependent −80 °C. The clinical information of patients are in Sup- manner, and promotes migration/invasion and metas- plementary Tables 1 and 2. Cervical cancer cells and other tasis in cervical cancer. cancer cells used in this study were obtained from the Tissue inhibitors of metalloproteinases (TIMPs) reg- American Type Culture Collection (ATCC; Manassas, ulate a vast range of the matrix and cell surface proteins, VA, USA). generating prominently effects on cancer metastasis and colonization49,50. Some studies have delineated the pro- Cell culture and transfection moter of TIMP3 is hypermethylated, which result into The human cervical cancer cell lines HeLa and C33A were repression of TIMP3 expression to facilitate the invasion maintained in the RPMI 1640 and MEMα, respectively, – and metastasis in various tumors51 53. More researchers supplemented with 10% FBS, 100 IU/ml penicillin, and focus on the DNA methylation of TIMP3 in cervical 100 μg/ml streptomycin, and incubated at 37 °C in a humi- 54–56 cancer . In this study, we found that TIMP3 was dified incubator supplemented with 5% CO2.Transfections targeted and downregulated by miR-G-10, which pro- were performed with Lipofectamine 2000TM reagent (Invi- motes invasion, adhesion, and metastasis in cervical can- trogen) according to the manufacturer’sprotocol. cer cells. The PI3K/AKT signaling pathway regulates the progress of cell cycle, cellular proliferation, and metabo- Deep sequencing and bioinformatics analysis lism57. Here, our data indicate that miR-G-10 upregulates Flag-RIP assay was used to extract RNA from HeLa cell PIK3R3 expression in cervical cancer cells. Upregulated transfected with Flag-GRSF1 according to the manu- PIK3R3 activates the PI3K/AKT/NF-κB pathway, and facturer’s protocol. The samples were qualified and sent promotes anoikis resistance to contribute the metastasis to Beijing Novogene Company for sequencing, and 18–30 of cervical cancer cells. These results indicate that miR-G- nt sequences were identified. The total read counts of 10 might function as an oncogene in cervical cancer. deep sequence showed that miR-G-10 is the most abun- In conclusion, a novel and highly expressed miRNA, dant with 617 reads among the 12 novel miRNAs. The miR-G-10, was identified and promotes metastasis in precursor of miR-G-10 is located at the Chromosome 5 cervical cancer cells. Furthermore, we revealed that miR- (136129333, 136129440), which is the noncoding region G-10 represses TIMP3 expression with the AGO2 com- of human SMAD family member 5 (SMAD5). Accord- plex, but activates PIK3R3 expression by binding to its 3′ ingly, we have uploaded the sequence information into UTR in a GRSF1-dependent manner, which linked to the NCBI gene database and gained the Genbank accession PI3K/AKT/NF-κB and TIMP3/ADAM17/MMP9 path- number: Homo MN153037. ways to contribute the oncogenic activity of miR-G-10. Moreover, miR-G-10 has a significant inhibitory effect on Plasmid construction β-integrin, which accelerate the enhancement of miR-G- To overexpress miR-G-10, the primary miR-G-10 was 10 on the metastatic ability in cervical cancer cells amplified from genomic DNA of HeLa cell and cloned (Fig. 7g). These findings might shed light on mechanism into the pcDNA3 vector between the KpnI and XhoI sites. underlying miRNA facilitating tumorigenesis and may The 3′ UTRs of TIMP3 and PIK3R1 that contain the miR- provide a new biomarker for cervical cancer. G-10-binding sites and mutant 3′ UTR fragments with mutant miR-G-10-binding sites were obtained from Materials and methods cDNA, and inserted these into the pcDNA3/EGFP vector. Materials The 3′ UTRs were put at the downstream of EGFP ORF. All clinical samples of human cervical tissues and serum For silencing miR-G-10 (anti-miR-G-10), TIMP3 (shR- were obtained from Tianjin Central Hospital of Gyne- TIMP3), and PIK3R3 (shR-PIK3R3), we constructed the cology & Obstetrics. Written informed consent was shRNA plasmid by annealing double-strand hairpin obtained from all enrolled patients, and all relevant cDNA and cloning the annealed oligonucleotides into the investigations were performed according to the principles pSilencer 2.1-neo vector. The gene expressing TIMP3 and

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Fig. 7 (See legend on next page.)

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(see figure on previous page) Fig. 7 miR-G-10 regulates NF-κB and extracellular matrix pathways in cervical cancer cells. a, b Western blot assay showed the pAKT and AKT protein level after transfection with PIK3R3, shR-PIK3R3, miR-G-10, and anti-miR-G-10. c–f HeLa cells were transfected with the indicated combinations of miR-G-10, anti-miR-G-10, PIK3R3, and shR-PIK3R3. After 48 h, cells were harvested, and NF-κB1 was detected by western blotting and immunofluorescent staining. n-NF-κB1: nuclear NF-κB, CENPA as the control group; c-NF-κB: cytoplasmic NF-κB, GAPDH as the control group. Scale bar: 10 μm. g The diagram model by which miR-G-10 upregulates PIK3R3 in the GRSF1 complex and downregulates TIMP3 in an AGO2 pathway to activate the AKT/NF-kB and MMP9/ADAM17 pathways, respectively, which promote the resistance of anoikis and malignancy in cervical cancer cells. All of the experiments were repeated three times. *P < 0.05; **P < 0.01; ***P < 0.001. NS not significant

PIK3R3 were amplified from the cDNA of HeLa cells, and Colony-formation, transwell, and wound-healing assay were cloned into the pCD3/flag tag vectors. All primers The details of Cell biology assays were performed as for PCR amplification are provided in Supplementary previously described14,16. Table 3. Immunofluorescent staining RT-qPCR Cells were seeded in 24-well plates and transfected with The total RNAs of HeLa and C33A were extracted using the indicated plasmids. Cells were then fixed with 4% the TRIzol regent, and reversely transcribed into cDNA paraformaldehyde, permeabilized with 0.05% TritonX with M-MLV reverse transcriptase (Promega, Madison, 100, and blocked with 10% donkey serum. After incu- WI, USA) according to the manufacturer’s protocol. The bating overnight with primary antibodies, secondary SYBR PremixEx Taq Kit (TaKaRa, Shiga, Japan) was used fluorescent antibodies were added, and DAPI was used for according to the manufacturer’s instructions, and RT- nuclear staining. Samples were imaged through a confocal qPCR was performed and analyzed using the iQ5 Detec- microscope. tion System (Bio-Rad, Hercules, CA, USA). All the primers (Supplementary Table 3) were purchased from RNA immunoprecipitation assay AuGCT, Inc. (Beijing, China). RIP assay was carried out following the method described by Christoph et al.19. EGFP reporter assay Cells were seeded in 48-well plates 24 h before trans- Cell–matrix adhesion assay fection, and then co-transfected with miR-G-10 or anti- In brief, 96-well plates were coated overnight with miR-G-10 and TIMP3-3′ UTR-EGFP, PIK3R3-3′ UTR- 10 μg/ml FN (Solarbio, Shanghai, China) at 4 °C and were EGFP, or TIMP3-3′ UTR (mut)-EGFP, PIK3R3-3′ UTR blocked with 1% (w/v) bovine serum albumin 1 h at 37 °C. (mut)-EGFP. The RFP expression vector, pDsRed2-N1 Cells were suspended in complete medium with RGDfv (Clontech, Mountain View, CA, USA), for normalization. (100 μM) and seeded on the 96-well plates at a density of Two days after transfection, cells were lysed with RIPA 2×104 per well in triplicate, allowed to adhere at 37 °C for and measured with fluorescence spectrophotometer at least 10 min, and were then washed three times with (F4500, Hitachi, Tokyo, Japan). phosphate-buffered saline. The cells were fixed with 4% (w/v) paraformaldehyde for 30 min, stained with 0.5% Western blotting analysis (w/v) crystal violet for 10 min, and then the attached cells First, protein samples were obtained by lysing cervical lysed with 2% SDS for 15 min; absorbance at 620 nm was cancer cell with RIPA lysis buffer for SDS-PAGE. The then measured. following antibodies were employed for immunoblotting: TIMP3, PIK3R3, MMP9, ADAM17, E-cadherin, vimentin, In vivo metastasis assay GAPDH, CENPA, and AKT were purchased from Tianjin Six-week-old nude mice Bal b/c were randomly assigned Saier Biotech (Tianjin, China). The secondary goat anti- to two groups (n = 4). The HeLa cells were transfected rabbit antibody was obtained from Sigma-Aldrich (St with miR-G-10 or control vector pcDNA3. Overall, 1 × Louis, MO, USA). 106 transfected cells were suspended by 100 μl of PBS and were injected into the veins of the tails. After 6–8 weeks, MTT assay the mice were killed, and the lungs were removed for At 24 h post transfection, HeLa and C33A were seeded H&E staining. in 96-well plates at 5000 cells per well. Cell viability was determined by MTT assay at 24, 48, and 72 h after Statistical analysis transfection. The absorbance at 490 nm was measured All analyses were performed using SPSS 19 for Windows using a Quant Universal Microplate Spectrophotometer (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 5 for (BioTek, Winooski, VT, USA). Windows (GraphPad Software Inc., San Diego, CA, USA).

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