PIK3R1 negatively regulates the epithelial-mesenchymal transition and stem-like phenotype of renal cancer cells through the AKT/GSK3β/CTNNB1 signaling pathway

The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway has been identified as an important pathway in renal cell carcinoma (RCC). We have reported a nonsense mutation in PIK3R1, which encodes the regulatory subunit of PI3K, in a metastatic RCC (mRCC), while the mutation was absent in the corresponding primary RCC (pRCC). To identify the function of PIK3R1 in RCC, we examined its expression in normal kidney, pRCC and mRCC by immunohistochemistry and real-time polymerase chain reaction. The expression of PIK3R1 significantly decreased in pRCC and was further reduced in mRCC compared with normal tissue. Besides, its expression levels were negatively correlated with T-category of tumor stage. Additionally, 786-O and A-704 cells with PIK3R1 depletion introduced by CRISPR/Cas9 system displayed enhanced proliferation, migration and epithelial-mesenchymal transition (EMT), and acquired a stem-like phenotype. Moreover, the PIK3R1 depletion promoted the phosphorylation of AKT in the cells. The knockdown of AKT by shRNA reduced p-GSK3β and CTNNB1 expression in the cells, while the depletion of CTNNB1 impaired stem-like phenotype of the cells. Overall, PIK3R1 down-regulation in RCC promotes propagation, migration, EMT and stem-like phenotype in renal cancer cells through the AKT/GSK3β/CTNNB1 pathway, and may contribute to progression and metastasis of RCC.

plasma membrane and generates phosphatidylinositol-3-phosphate (PIP3) by phosphorylating phosphatidylinositol 4, 5-bisphosphate 8 . Through a pleckstrin homology domain, AKT binds to PIP3 and is phosphorylated to pAKT 8 . Class IA PI3Ks are heterodimers that consist of a catalytic subunit (p110a, p110b and p110d) and a regulatory subunit (p85a, p55a, p50a, p85b, and p55c) 9 . The catalytic subunit p110a is encoded by PIK3CA, while the regulatory subunit p85a is encoded by PIK3R1 9 . The PI3K/AKT pathway regulates many aspects of cellular functions, including cell growth, proliferation, translation and survival, and is also involved in pathological conditions 6 . Deregulation of the pathway can lead to disturbance of cell growth, proliferation and survival, thus resulting in growth advantage and metastatic competence of cancer cells 10 .
The alteration of PI3K/AKT pathway has been found in a broad spectrum of cancers 9 . Members of the pathway such as PIK3CA and PTEN are frequently altered in RCC 2 . Since the pathway plays an important role in RCC pathogenesis 2 , it has been showing a great promise for molecularly targeted treatment of RCC 6,9 . However, only a small number of patients benefit from single-agent PI3K targeted therapy11. The related mechanism of unsatisfied effect of PI3K targeted therapy remains to be clarified 11 . Can, in addition to PIK3CA and PTEN, other member of the pathway play a role and be a target of therapy in RCC?
Accumulating studies have showed an important role of PIK3R1 in human carcinogenesis 8,[12][13][14] . PIK3R1 has been reported as an oncogene in ovarian and colon tumors 15 , whereas it has been shown as a tumor suppressor in hepatocellular carcinomas 16 . The underexpression of PIK3R1 has been reported to be associated with poor prognosis of breast cancers 17 . A missense mutation of PIK3R1 which resulted in decrease of PIK3R1 expression has also been strongly linked with colon cancers 18 . We have reported a nonsense mutation in PIK3R1 in an mRCC, while the mutation was absent in the corresponding primary renal cell carcinoma (pRCC) 14 . Therefore, we hypothesize that the downregulation of PIK3R1 may confer renal cancer cells a selective advantage to translocate, colonize and develop as mRCC.
We speculate that ectopic expression of PIK3R1 may be associated with progression and metastasis of RCC. To examine our hypothesis, we firstly analyzed the expression of PIK3R1 in RCC including both pRCC and mRCC by immunohistochemistry (IHC) and real-time polymerase chain reaction (RT-PCR). We discovered that the expression of PIK3R1 in RCC negatively correlated with tumor progression and metastasis. In addition, we induced deletion mutations of PIK3R1 in renal cancer cell lines (786-O and A-704 cell lines) using a CRISPR/Cas9 system to achieve haploid knockout of PIK3R1 which significantly decreased the expression of P85a. The mutated renal cancer cells displayed increased abilities of colony formation, tumor formation, migration, epithelial-mesenchymal transition and oncosphere formation. Thus, our current study demonstrates that the downregulation of PIK3R1 contributes to progression and metastasis of RCC.

Results
Downregulation of PIK3R1 correlates with progression and metastasis of RCC. In order to examine the expression of PIK3R1 in RCC, the protein expression of PIK3R1 in normal kidney (n 5 13), pRCC (n 5 13) and mRCC (n 5 21) was determined by IHC. As shown in Fig. 1a, normal kidney tissues displayed high level of PIK3R1 expression, whereas the expression of PIK3R1 was decreased in pRCC and was further reduced to a lower level in mRCC. The mRNA expression of PIK3R1 was then determined by real-time polymerase chain reaction (RT-PCR). Compared with normal kidney tissue group, the mRNA expression of PIK3R1 was significantly decreased in RCC group (n 5 18) (Fig. 1b). The epithelial-mesenchymal transition (EMT) is considered to be crucial to tumor progression and metastasis, in which NCAD is the hallmark of EMT 19,20 . To determine whether the downregulation of PIK3R1 could affect the expression of NCAD, the mRNA expression of NCAD was examined, and data showed that the expression of NCAD had a negative correlation with the mRNA expression of PIK3R1 (Correlation 5 0.6929, P 5 0.0014) (Fig. 1c). Additionally, the mRNA expression of PIK3R1 negatively correlated with the T category of tumor, although there was no significant difference among different grades (Fig. 1d). These data suggest that the downregulation of PIK3R1 in RCCs correlates with their progression and metastasis.
Haploid knockout of PIK3R1 by CRISPR/Cas9 promotes tumor proliferation. In order to reveal the functional influence of PIK3R1 depletion, a CRISPR/Cas9 strategy which was designed according to the nonsense mutation reported previously 14 , was applied to induce a haploid deletion mutation in order to achieve haploid knockout of PIK3R1. Firstly, we analyzed the expression levels of PIK3R1 in normal renal cell line (HK2) and RCC cell lines (786-O, A-498, A-704, and ACHN) by using RT-PCR. 786-O and A-704 cell lines showed higher expression of PIK3R1 compared with that of HK2 cell line ( Supplementary Fig. 1). Therefore, we performed depletion of PIK3R1 in 786-O and A-704 cell lines. We also confirmed the sequence character of chr5: 67576819 in PIK3R1 in 786-O and A-704 cell lines with Sanger sequencing. The 786-O and A-704 cell lines harbored the wild type (WT) sequence of chr5: 67576819C in PIK3R1 (Fig. 2a).
To determine the effects of haploid knockout of PIK3R1 on the growth of renal cancer cells, we carried out colony formation assays for the wild type and mutant cells. 786 and A-704 mutated cells displayed enhanced colony formation capability under the condition of separated single cells than that of 786-O and A-704 WT cells (Fig. 2c). In order to evaluate the tumor formation capability of the WT and muted cells, they were respectively inoculated subcutaneously into NOD/SCID mice to carry out tumor formation assays. The mutated cells formed bigger tumors than those of the wild type cells in NOD/SCID mice (Fig. 2d).
Haploid knockout of PIK3R1 promotes renal cancer cells migration and EMT in vitro. To confirm the effects of haploid knockout of PIK3R1 on the migration of renal cancer cells, woundhealing and transwell assays for 786-O, 786-mut1, 786-mut2, A-704, A-704-mut1, and A-704-mut2 cells were carried out. Eighteen hours after artificial wounding, 786-O and A-704 WT cells demonstrated slight migration, whereas 786 and A-704 mutated cells showed enhanced migration ability and healed more than half of the wound (Fig. 3a). The 786 and A-704 mutated cells showed higher ability of migration in the transwell assay than that of 786-O and A-704 WT cells (Fig. 3b).
Additionally, 786 and A-704 mutated cells displayed a mesenchymal morphology 21 that was different from the epithelial morphology of 786-O and A-704 WT cells, indicating that 786 and A-704 mutated cells might undergo EMT (Fig. 3c). In the wild type and mutant cells, the expression of EMT related gene was detected by RT-PCR and WB 20  WT cells, in 786 and A-704 mutated cells with the downregulation of PIK3R1, the mRNA expression of ECAD was decreased, whereas the mRNA expression of NCAD, VIM and ZEB1 was increased (Fig. 3d). These observed changes were also found in the protein levels of ECAD (P , 0.01; P , 0.01), NCAD (P , 0.001; P , 0.05), VIM (P , 0.01; P , 0.05) and ZEB1 (P , 0.05; P , 0.05) in 786 and A-704 mutated cells respectively (Fig. 3d). However, no differences of CD44 and POU5F1 were observed between the wild type and mutant cells (Fig. 3d).
In order to confirm the function of PIK3R1 in renal cancer cells, we analyzed the expression of ECAD, NCAD, VIM, SNAIL, and TWIST in normal renal cell (HK2) and RCC cell lines (786-O, A498, A704, and ACHN) with RT-PCR. ACHN cell line showed higher expression of NCAD and VIM, compared to HK2 cell line ( Supplementary Fig. 1). Therefore, we performed overexpression of PIK3R1 in ACHN cells. Compared with ACHN-vec cells, the mRNA expression of PIK3R1 and ECAD was increased in ACHN-PIK3R1 cells, whereas the expression of NCAD, VIM and ZEB1 was decreased ( Supplementary Fig. 3a). These changes were also observed in the protein levels of PIK3R1 (P , 0.01), ECAD (P , 0.05), NCAD (P , 0.01), VIM (P , 0.05), and ZEB1 (P , 0.05) in ACHN-PIK3R1 cells ( Supplementary Fig. 3b). In addition, ACHN-PIK3R1 cells displayed a epithelial morphology 21 that was different from the mesenchymal morphology of ACHN-vec cells ( Supplementary Fig.  3c). These results indicated that ACHN-PIK3R1 cells might undergo mesenchymal-epithelial transition (MET). Taken together, PIK3R1 negatively regulated renal cancer cell migration and EMT in vitro.
Haploid knockout of PIK3R1 promotes a cancer stem cell phenotype. Cancer stem cells (CSCs) are defined as a subpopulation in tumors which harbors selfrenew, differentiation and serial tumor formation ability 22 . Cancer cell lines contain cancer stem-like cells which can form cell spheres under sphere-forming conditions 23 . To evaluate whether the reduction of PIK3R1 influences the cancer stem cell phenotype of renal cancer cells, cell sphere formation assays of 786-O, 786-mut1, 786-mut2, A-704, A-704-mut1, and A-704-mut2 cells were performed. After two weeks of cultivation, 786 and A-704 mutated cells developed more and bigger spheres than that of 786-O and A-704 WT cells under the medium supplemented with 20 ng/mL EGF, FGF, N2, and B27 (Fig. 4a). CSCs have been identified based on expression of various markers such as CD44, CD133 and CXCR4 [24][25][26] . By using anti-CD44 antibodies, anti-CD133 antibodies and anti-CXCR4 antibodies we observed that 786 and A-704 mutated cells comprised more CD44 1 , CD133 1 and CXCR4 1 subset, respectively, compared with 786-O and A-704 WT cells (Fig. 4b).
The gold standard assay to assess CSCs potential is the transplantation of limiting dilutions of highly purified prospectively identified cancer cell populations into immunodeficient mice to assess their ability to form tumors 27,28 . Transplantation of different dilutions of the wild type cells and PIK3R1 mutated cells showed that the mutated cells had a much greater ability to initiate tumors and increasing percentage of CSCs compared with the WT cells (Fig. 4c).
Haploid knockout of PIK3R1 activates WNT/b-catenin pathway dependent on the phosphorylation of AKT. In order to uncover the The TNM cancer staging system was designed to gauge the extent of cancer in a patient's body. T describes the size of the tumor and whether it has invaded nearby tissue, N describes regional lymph nodes that are involved, and M describes distant metastasis (spread of cancer from one body part to another). Grading classification for RCC was based on the Fuhrman grading system. Data are presented as mean 6 SD. * P , 0.05, ** P , 0.01. www.nature.com/scientificreports SCIENTIFIC REPORTS | 5 : 8997 | DOI: 10.1038/srep08997 stem cell signaling pathway activated by the downregulation of PIK3R1, the mRNA level of CTNNB1, HES1, GLI1, and NANOG were analyzed in 786-O, 786-mut1, 786-mut2, A-704, A-704-mut1, and A-704-mut2 cells by RT-PCR. With the downregulation of PIK3R1 in 786 and A-704 mutated cells, the mRNA expression of CTNNB1 was higher than that in 786-O and A-704 WT cells (Fig. 5a). The protein levels of CTNNB1 were also increased in 786 and A-704 mutated cells respectively (P , 0.01; P , 0.05) (Fig. 5d) There were no significant differences in the mRNA expression of HES1 and GLI1 between the wild type and mutated cells (Fig. 5a). Though there were no significant differences in the NANOG mRNA expression between 786-O WT and 786 mutated cells, significant difference was observed between A-704 WT and A-704 mutated cells (Fig. 5a).

Discussion
Over the past 20 years, the functional role of the PI3K/AKT pathway in tumorigenesis has been reported by numerous studies 31 . Members of the pathway such as PIK3CA, AKT and PTEN, are frequently altered, and cause the activation of the pathway in RCC 2,14 . As a regulatory subunit of PI3Ks, PIK3R1 alteration is involved in carcinogenesis in a variety of cancers 9 , whereas the alteration of PIK3R1 in RCC has been rarely reported 14 . In this study, we report that PIK3R1 expression is reduced in RCC, and that the reduction of PIK3R1 can result in EMT, enhance migration ability, and promote colony formation, tumor formation and cancer stem cell phenotype in renal cancer cells.
Our data showed that PIK3R1 expression was reduced in RCC, especially in advanced and metastatic RCC, and the downregulation of PIK3R1 correlated with advanced or metastatic RCC. These results are in consistence with discoveries in hepatocellular and colon cancers 16,18 , indicating that the reduction of PIK3R1 expression may acquired tumorigenicity in RCC, and thus supporting the view that PIK3R1 may function as a potential cancer suppressor, and that the downregulation of PIK3R1 may promote progression and metastasis of RCC.
We then tested the role of PIK3R1 in renal cancer cell lines. We observed that the haploid deletion mutations of PIK3R1 by CRISPR/Cas9 system led to the haploid knockout and downregulation of PIK3R1, and generated cell lines with the insufficiency of PIK3R1. The binding of p110a to p85a was decreased in 786 and A704 mutated cells with the reduced expression of PIK3R1. We also showed that the downregulation of PIK3R1 functionally activated the PI3K/AKT pathway, as evidenced by the enhanced phosphorylation of AKT in Ser473 residue. The activation of the pathway may be due to the combined effect of several factors. The p85 regulatory subunit of PI3K is necessary to stabilize and recruit p110 catalytic subunit onto cellar membrane 13 . However, monomeric p85 is in competition with p85-p110 heterodimers for binding to insulin receptor substrate-1 (IRS-1) to form a non-signaling cytosolic protein complex 32 . The insufficiency of PIK3R1 expression would primarily result in the reduction of monomeric p85, which would suppress the negative regulation of IRS-1/PI3K pathway by p85, thus leading to the hyperactivation of PI3K signaling axis 32 . PIK3R1 has also been shown to have a positive regulatory effect on tumor suppressor PTEN, which negatively regulates the PI3K/AKT pathway 16 . Therefore, the downregulation of PIK3R1 leads to the inhibition of PTEN function that reduces the breakdown of PIP3, and activates the downstream of the PI3K/AKT signaling axis 16 .
Our data show that the reduction of PIK3R1 expression increases the motility and migration capacity of renal cancer cells. A growing body of evidence indicates that the PI3K/AKT pathway is involved in the migratory process of cells, including metastatic cancer cells 33 . Activated AKT mediates cell polarity and reorganizes cytoskeleton, thus regulates the contraction of the cellular body and facilitates the migration of cells 33 . AKT-mediated phosphorylation activates components closely related to the cellular filaments which are important for cytoskeleton dynamics 34 . In our present study, the expression of VIM was elated in cells with the downregulation of PIK3R1. Vimentin, which is phosphorylated and protected by AKT from degradation 35 , is substantially up-regulated when cells are highly motile, especially during EMT. It can promote the migration of different type of cells such as cancer cells 35 .
Interestingly, spindle-shaped morphology of mesenchymal cells has been observed in cells with the down-expression of PIK3R1. EMT is conducive to the survival and spread of cancer cells 36 . The activation of AKT induces EMT and contributes to migration of squamous cell carcinoma lines 37 . Evidences are emerging that the action of AKT is related with EMT-inducing transcription factor 38 . The hallmarks of EMT include decreased expression of epithelial marker, such as ECAD, and a simultaneous increased expression of mesenchymal markers, such as NCAD, VIM, CD44, ZEB1, and POU5F1 20 . In our study, the expression of NCAD, VIM and ZEB1 was elated in the cells with the downregulation of PIK3R1, whereas the expression of ECAD was decreased in these cells. The activated AKT positively regulates the expression of NCAD 33 , and the inhibition of the PI3K/AKT signaling inhibits the expression of NCAD 39 . N-cadherin encoded by NCAD is enriched in cellular protrusions and plays a key role in cell migration 33 . Altogether, through the activation of the PI3K/AKT pathway which phosphorylates several cytoskeleton-regulating and EMT-activating proteins, the downregulation of PIK3R1 promotes migration and EMT in renal cancer cells.
CSCs are defined as cells which are able to produce a new tumor 22 , and regarded as a source of tumor progression and metastasis 22 .

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Cancer cell lines contain cancer stem-like cells 23 . In our current study, we observed that the down-expression of PIK3R1 promoted the colony formation, cell sphere and tumor formation of renal cancer cells and increased the expression of CTNNB1. Our data are consistent with the study by Zhou et al, who reported that the CSC viability and maintenance of breast cancer stem-like cells required the activation of the PI3K/AKT signaling axis 40 . It has been suggested that the PI3K/AKT pathway is critical to maintain prostate cancers stem-like cells 41 . Signaling pathways including the Hedgehog/GLI1, WNT/CTNNB1, NANOG/OCT4, and Notch/HES1 are activated in CSCs and contribute to the function of CSCs 42 . We performed RT-PCR to reckon the expression of CTNNB1, HES1, GLI1, and NANOG. Interestingly, no significant change was observed in the expression of HES1, GLI1 and NANOG, whereas the expression of CTNNB1 was markedly increased, suggesting that CTNNB1 may be involved in increased renal cancer stem-like phenotype caused by the down-expression of PIK3R1.
Our present study demonstrates that the downregulation of PIK3R1 in RCC enhances stem cells expansion as a result of the activated PI3K/AKT signaling, involving the WNT/CTNNB1 pathway. The PI3K/AKT pathway has always been ascribed to the proliferation and self-renewal of cancer stem-like cells 29 . However, under certain conditions, such as ectopic gene expression, cancer stem-like cells can also be transformed from cells lacking cancer stem-like phenotype 43 . The activation of the PI3K/AKT pathway has been reported to induce differentiated cells to transform into highly tumorigenic and cancer stem-like cells 44 . In our study, after the depletion of AKT, the expression of p-GSK3b and CTNNB1 was decreased in 786-mut1 shAKT and A-704-mut2 shAKT cells. Therefore, in consistence with previous studies, our study suggests that the PI3K-AKT and WNT/CTNNB1 pathways converge on GSK3b in renal cancer cells 29 . The WNT/CTNNB1 signaling is involved in regulating the various types of stem cells 29 . In present study, the depletion of CTNNB1 in renal cancer cells decreased the cancer stem-like phenotype of these cells. Activated AKT can block the kinase activity of GSK3b by phosphorylating GSK3b 30 , resulting in the accumulation of b-catenin which transforms differentiated cancer cells to cancer stem-like cells 29,30 . Therefore, the PI3K/AKT and WNT/CTNNB1 pathway work together as a PI3K/AKT/GSK3b/CTNNB1 pathway which induces differentiated renal cancer cells to transform into a more cancer stem-like cells 29 .
To our knowledge, this study is the first report of the functional identification of PIK3R1 in RCC. The important discovery of our study is that PIK3R1 is down expressed in RCC, especially in advanced and metastatic RCC. We observed that the downregulation of PIK3R1 resulted in EMT, increased migration, colony formation, cell sphere, and tumor formation in renal cancer cells, suggesting that the reduction of PIK3R1 expression confers renal cancer cells a selective advantage to translocate, colonize and develop as mRCC, and that PIK3R1 may be involved in the progression and metastasis of RCC through the PI3K/AKT/GSK3b/CTNNB1 pathway. Since the reduction of PIK3R1 expression and activating alterations in PIK3CA can simultaneously be involved in RCC, conformational changes may result from these alterations. These changes would eliminate the repression of PIK3CA caused by PIK3R1 32 . Therefore, the exploitation of agents that can restore the repression regulation of PIK3R1 and inhibit the expression of PIK3CA, may be a new strategy to treat RCC.
In summary, our study suggests that PIK3R1 is down-regulated in RCC, especially in advanced and metastatic RCC. The downregulation of PIK3R1 promotes EMT, migration, and cancer stem cell phenotype in renal cancer cells and may contribute to the progression and metastasis of RCC by activating the PI3K/AKT/GSK3b/ CTNNB1 pathway. Increasing or restoring expression of PIK3R1 may be a novel treatment strategy for RCC. Immunohistochemistry. Sections (4 mm) were deparaffinized and rehydrated. After antigen retrieval, these sections were treated with 3% H 2 O 2 solution, incubated with 8% bovine serum albumin for 30 min and primary antibody at 4uC overnight, then incubated with corresponding secondary antibody, and subsequently stained with DAB kit (ZSGB Bio). The nucleus was counterstained with hematoxylin. The overall staining for PIK3R1 was measured by the multiplication of staining percentage (0%-100%) and staining intensity on a numerical scale (none 5 1, weak 5 2, moderate 5 3, strong 5 4), resulting in an overall product score.
Real-Time PCR analysis of PIK3R1 expression. Total RNA was extracted using RNA isolation kit (Tiangen Biotech) and subjected to cDNA synthesis using PrimeScript RT reagent Kit (Takara Bio). The cDNA was then used for the evaluation of the relative mRNA levels of the indentified gene, running in an ABI 7300 analyzer (Applied Biosystems). SYBR Green I (Tiangen Biotech) was used as the fluorescent probe. Primer sequences for RT-PCR are listed in the supplementary table 3. The relative expression levels of the target genes were referred to as a housekeeping gene, GAPDH.
PIK3R1 knockout by CRISPR/Cas9. The knockout of PIK3R1 in 786-O and A-704 cell lines was achieved by CRISPR/Case9 system. One 23-base sgRNA (59-AATGAACGACAGCCTGCACCAGG-39) was designed to the target site (g.chr5: 67576819) in the PIK3R1 (Gene ID: 5295). The U6-gRNA expression cassette including U6 promoter and 183 bp sgRNA tails was synthesized (Invitrogen) and inserted into pCEP4 vector (Invitrogen) by KpnI and XhoI. The sgRNA was synthesized (Invitrogen) and inserted into two AarI site between U6 promoter and sgRNA tails to form the pCEP4-U6-gRNA expression vector (Supplementary Figure  2). The sequence of Cas9 was consistent with previous reports 46,47 . The T7 promoter was added upstream of the Cas9 sequence. All the sequences were synthesized (Invitrogen) and ligated to the pMD-18T-CMV-MCS-BGPA by NheI/AflII to form CMV-Cas9 expression vector (Supplementary Figure 2). 786-O and A-704 cells were transfected according to Lipo-fectamine 2000 (Invitrogen) protocol. Briefly, Cells were transfected at 60% confluency in 12 well with 500 ng of pCEP4-U6-gRNA expression vector, 500 ng of CMV-Cas9 expression vector and 3 mL Lipo-fectamine 2000. After G418 (500 ng/mL) selection for 10 days, G418-resistant colonies were picked and cultured in 24 well plates. Genomic DNA was extracted with E. Z.N.A. Tissue DNA Kit (OMEGA). Targeted cleavage was measured by PCR amplification (forward primer: 59 CTCATCAGTATTGGC-TTACGCTT 39 and reverse primer: 59 GGTTGTTTAGACTTTCCACGGTA 39) and T7 E1 assay. The fragments were determined by Sanger sequencing. To better verify the mutations (insertions and deletions), the PCR product of mutated colonies was then ligated to pMD-18T vector (Takara Bio). Individual mutation and mutational spectra were obtained from Sanger sequence. Cell clones with haploid deletion www.nature.com/scientificreports mutation of PIK3R1 were selected for evaluating the function of PIK3R1 in renal cancer cells.
Western blot and immunoprecipitation (IP). Cells were lysed with radioimmunoprecipitation assay buffer (Beyotime Biotech). Protein was determined, separated and transferred to polyvinylidene difluoride membranes (Pall Life Sciences). The membranes were blocked, then probed with corresponding primary antibody and HRP-conjugated secondary antibodies (Beyotime Biotech), and detected using BeyoECL PLUS Kit (Beyotime Biotech).
For immunoprecipitation assays, cell lysates were incubated with the PIK3R1 antibody overnight and precipitated with protein A-Sepharose 4B beads (Invitrogen) at 4uC for 4 h. The immunoprecipitated proteins were washed, separated on SDS-PAGE, transferred onto a PVDF membrane, and followed by western blot analysis.
Colony formation assay. Briefly, single cells were seeded onto 6-well plates (Corning) at a density of 1000 cells/well and then incubated at 37uC. After two weeks of culture, colonies were stained with 0.5% crystal violet solution for 30 min at room temperature. The colonies from three replicate wells were counted, and pictures were captured digitally.
Generation of xenografts. For generation of xenografts, different dilutions of cells were injected with Matrigel (BD Biosciences) subcutaneously into the backs of NOD/ SCID mice. The volume of xenografts was serially measured. The mice were sacrificed after 30 days. Animal work was carried out in compliance with the ethical regulations approved by the Animal Care Committee, Southern medical University.
Wound healing assay. Briefly, 4 3 10 5 cells were seeded into a 24 well plate. After overnight incubation, monolayer cells grew to confluency. Artificial wounds were introduced with the P200 pipette tip. The pictures of the wounded area were taken immediately (time 0 h), and at 18 h with an inverted microscope (Olympus Corp). The whole assay was repeated three times.
Transwell Assay. Cells were respectively harvested, suspended and added to the upper compartment of transwell inserts (8 mm pore size; Corning). The lower chambers were filled with RPMI-1640 supplemented with 10% FBS. After incubation for 16 hr, cells that had not migrated from the upper chamber were scraped away. The cells that migrated to the lower surface of each membrane were stained and counted under a light microscope in in five fields/well. The whole assay was repeated three times.
Cell labeling and flow cytometry analyzing. Cells were harvested and washed twice in ice-cold PBS. Then cancer cells were stained with FITC-conjugated anti-CD133 antibody, PE-conjugated anti-CD44 antibody and FITC-conjugated anti-CXCR4 antibody, respectively, for 30 min on ice. After washing with PBS, the labeled cells were analyzed by flow cytometry (BD FACSAria III).
Statistical analysis. Student's t-test was used to comparing the means of two samples. Linear correlation analysis was used to determine relationship between PIK3R1 and NCAD mRNA expression. A value of p less than 0.05 (* P , 0.05, ** P , 0.01) was regarded statistically significant.