Abstract

   There are some controversies about the involvement of microRNA
   (miR)-19a-3p in hepatocellular carcinoma (HCC) biology, even though
   many studies have shown that it plays an important role in cancer. In
   this study, we found that miR-19a-3p is usually overexpressed in HCC
   tissues compared with corresponding peritumorous tissues, and its
   expression was associated with tumor size and poor overall survival.
   MiR-19a-3p promoted cell proliferation significantly, and more cells
   were found in the S phase. In vivo, miR-19a-3p promoted liver tumor
   growth, and more HCC cells were found in the active cell cycle.
   Sequencing and bioinformatics analysis predicted that PIK3IP1 is a
   likely target gene of miR-19a-3p, and we next confirmed it by
   luciferase and rescue assays. Altogether, our data showed an important
   role of PIK3IP1 downregulation by miR-19a-3p in HCC progression, and
   the miR-19a-3p-PIK3IP1-AKT pathway may be a potential therapeutic
   target.

   Keywords: microRNA-19a-3p, miR-19a-3p, phosphatidyl inositol 3-kinase
   interacting protein 1, PIK3IP1, hepatocellular carcinoma, cell growth

Introduction

   Hepatocellular carcinoma (HCC) is one of the most common cancer types
   [51]^1 and is the third leading cause of tumor-related deaths in China
   [52]^2^, [53]^3. Even though there have been continuous improvements in
   screening, diagnosis, and treatment over the past decades, an effective
   approach to control HCC progression is not available, except for
   surgical treatment. The 5-year survival rate of patients with liver
   cancer is only 18% when all stages are considered [54]^1. Therefore,
   understanding the molecular pathogenesis of HCC and identifying
   therapeutic targets are extremely urgent goals.

   MicroRNAs (miRNAs) are evolutionarily conserved small noncoding RNAs,
   which are known to play a major role in various biological phenomena,
   including cell differentiation, immune responses, and cancer [55]^4.
   Usually, miRNAs regulate gene expression at the post-transcriptional
   level by blocking mRNA translation or by causing degradation of target
   mRNAs by binding to their 3′-untranslated regions (3′-UTRs) [56]^5^,
   [57]^6. The miR-17-92 cluster is often upregulated, and individual
   members of the cluster may have distinct functions [58]^7. miR-19a-3p
   was identified as a key oncogenic miRNA of the miR-17-92 cluster
   [59]^8; it is dysregulated in many types of cancer, including gliomas,
   bladder cancer, breast cancer, and pancreatic, gastric, and laryngeal
   squamous cell carcinoma, whereas PPAR-α, PTEN, and RUNX3 have been
   identified as its target genes. miR-19a-3p is known to participate in
   tumor growth, cell survival, and metastasis [60]^9^, [61]^10. The
   involvement of miR-19a-3p in HCC is also well studied, but there are
   some controversial topics. Some studies have shown that miR-19a-3p is
   underexpressed in patients with recurrent HCC compared to the
   nonrecurrence group[62]^11. It inhibits cell proliferation and promotes
   more apoptosis in HCC [63]^12.These data indicate its tumor suppressor
   function but some studies that have been conducted show the opposite
   effects of miR-19a-3p in HCC. These studies indicate that miR-19a-3p is
   upregulated in an HCC tumors compared to respective peritumor adjacent
   tissues [64]^13 and it promotes tumor growth, metastasis, and
   chemoresistance [65]^14^, [66]^15. Therefore, the function of
   miR-19a-3p in HCC is still unclear and requires more studies.

   In the present study, we tested miR-19a-3p expression level in HCC
   specimens and analyzed the correlation of miR-19a-3p expression levels
   with outcomes (including recurrence) among 102 patients. We studied the
   participation of miR-19a-3p in HCC cell growth in a series of in vitro
   and in vivo experiments. To this end, we screened databases for
   putative target genes of miR-19a-3p using sequencing and bioinformatics
   analysis, and investigated the possible molecular mechanism. Our
   findings confirmed that miR-19a-3p promotes HCC cell proliferation and
   tumor growth and revealed a new target gene, PIK3IP1, thus providing
   new insights into the molecular functions of miR-19a-3p.

Materials and Methods

   Patients and HCC samples. Human liver tissues were obtained from
   surgically resected specimens of HCC from patients admitted at
   Zhongshan Hospital, Fudan University. All tumor specimens and matching
   adjacent nontumorous tissues were snap-frozen in liquid nitrogen after
   the surgical procedure and stored at -80 ºC. The 102 HCC patients were
   diagnosed with HCC between 2011 and 2012 and received no chemotherapy
   or radiotherapy before the resection; the details of these patients are
   described in Supplementary Table. Clinical data collection and
   follow-up procedures were performed according to a uniform guideline
   described previously [67]^16. Overall survival was defined as the time
   between the dates of surgery and death, and time of recurrence was
   defined as the interval between the dates of surgery and recurrence.
   The study protocol was approved by Zhongshan Hospital Ethics Committee,
   and informed consent was obtained from each patient in accordance with
   institutional review board protocols.

   Cell culture and transfection. Huh7 (HCC) cells were purchased from
   BeNa Culture Collection (Beijing, China). L02, BEL-7402, Hep3B cell
   lines were obtained from the cell bank of Chinese Academy of
   Sciences(Shanghai, China), MHCC97H cell lines was previously
   established in our laboratory. The cells were maintained in Dulbecco's
   modified Eagle's medium (DMEM) supplemented with 10% of fetal bovine
   serum. After 12 h of seeding, the cells were placed in a hypoxia
   workstation (In Vivo 200, Ruskinn Technology, Ltd., Bridgend, UK) for
   48 h at 1% O[2.] For a colony formation assay, 1000 cells were seeded
   in 35 mm dishes and incubated in DMEM with 10% of FBS. Two weeks later,
   the cells were fixed and stained with 0.1% crystal violet. To generate
   a lentivirus expressing miR-19a-3p, the precursor sequence was
   amplified by PCR (the amplicons carried overhangs) and was inserted
   into the MCS-SV40 expression vector. To knock down the miR-19a-3p
   expression in Huh7 cells, molecular sponges were used to decrease the
   miR-19a-3p level. To knock down the expression of PIK3IP1, the small
   interfering RNA (siRNA) and siRNA control were purchased from Thermo
   Fisher Scientific. For overexpression of PIK3IP1, the cDNA encoding
   PIK3IP1 was amplified by RT-PCR and cloned into vector GV358. Huh7
   cells were seeded in 6-well plates and incubated overnight, then
   transfected with the lentivirus according to the manufacturer's
   instructions.

   RNA extraction, cDNA, RT-PCR, mRNA sequence and western blotting. To
   analyze miRNA expression, total RNA was extracted with the miRNA
   Isolation Kit (Life Technologies, Carlsbad, CA), and complementary DNA
   was synthesized by the miRNA RT Assay (TaqMan). The miR-19a-3p (assay
   ID 002424, Thermo Fisher Scientific, Inc.) expression level was
   measured and normalized to small nuclear RNA U6 [68]^7. For mRNA
   analysis, RNA was extracted and purified using the TRIzol Reagent
   according to the manufacturer's protocol (Thermo Fisher Scientific,
   Inc.). cDNA was synthesized using random primers, and PCR was carried
   out with the SYBR Master Mix according to the manufacturer's protocol
   (Thermo Fisher Scientific, Inc.). GAPDH served as a control; the
   forward primer sequence was 5′-GGGGCTCTCCAGAACATCATCC-3′, and the
   reverse primer sequence was 5′-ACGCCTGCTTCACCACCTTCTT-3′. Mrna sequence
   was performed as previously [69]^17 and the data was uploaded to NCBI
   database ([70]GSE140148). The western blotting protocol was carried out
   following the method from our previous studies [71]^16. Specific
   antibodies against the following proteins were employed: cyclin A2
   (9869, Cell Signaling Technology, Danvers, MA, USA), Bak (3814s, Cell
   Signaling Technology), Bax (sc-7480, Santa Cruz Biotechnology, Inc.,
   USA), and PIK3IP1 (sc-365777, Santa Cruz Biotechnology, Inc.).

   In vivo experiment. Nude mice (4-6 weeks old, male) were kept under
   specific pathogen-free conditions. Huh7 (HCC) cells (5 × 10^6) were
   subcutaneously injected into the flanks of mice. After 4 weeks, the
   tumor volume reached ~1-2 cm^3. Next, these tumors were seeded into the
   liver parenchyma of nude mice under anesthesia after the abdomen was
   opened up. All the mice were killed 8 weeks later, following which the
   volume of tumors was calculated.

   Luciferase assay. PIK3IP1 with a potential miR-19a-3p-binding site or
   the corresponding mutant site was generated and ligated into luciferase
   reporter vector GV272. Cells (293T) were seeded in 24-well plates for
   24 h incubation. Next, 0.1 μg of a luciferase plasmid harboring the
   wild-type or mutant miR-19a-3p-binding site in the PIK3IP1 3′-UTR and
   0.4 μg of the miRNA-encoding plasmid were added into each well together
   with Lipofectamine 2000 in triplicate. Firefly and Renilla luciferase
   activities were measured consecutively using the Dual-Luciferase
   Reporter Assay System Kit (Cat. No. E1910, Promega) after 48 h of
   transfection. Renilla luciferase activity was used for normalization.

   Statistical analysis. Statistical analysis was performed in SPSS 22.0
   software for Windows. The chi-squared test was conducted to evaluate
   differences in categorical variables. Overall survival and time of
   recurrence were determined by the Kaplan-Meier method. A Cox regression
   analysis was performed for the multivariate analyses of prognostic
   factors. Statistical significance was set to p < 0.05.

Results

Elevated expression of miR-19a-3p promotes HCC progression and correlates
with poor outcomes

   A hypoxic microenvironment is an important characteristic of a tumor
   and is associated with tumor growth, metastasis, and poor outcomes.
   Previously, we have identified 58 differentially expressed miRNAs in
   HCC cell line Huh7 under hypoxia as compared with normoxia, and
   miRNA-mRNA network analysis revealed that miR-19a-3p is one of the key
   differentially expressed miRNAs [72]^17. Although miR-19a-3p has been
   studied in several cancers, the involvement of miR-19a-3p in HCC is
   still unclear. We initially examined miR-19a-3p expression in Huh7
   cells to confirm the miRNA array results, and they showed that ~3-fold
   upregulation of miR-19a-3p under hypoxic culture conditions occurred as
   compared to normoxic conditions, which is consistent with the
   microarray analysis (Figure [73]1A). It is known that there are many
   hypoxic areas in a tumor. We next measured the expression levels of
   miR-19a-3p in HCC tumor samples and their paired peritumor tissue
   samples by qRT-PCR. Notably, miR-19a-3p turned out to be markedly
   upregulated in tumor tissue samples compared with the paired peritumor
   tissue samples (Figure [74]1B). We then determined the correlation
   between miR-19a-3p expression and patients' outcomes (including tumor
   metastasis) among the 102 HCC cases. This analysis indicated that
   miR-19a-3p expression was positively correlated with tumor size and
   encapsulation ([75]Supplementary Table 1). Kaplan-Meier analysis
   indicated that high miR-19a-3p expression was associated with shorter
   overall survival (Figure [76]1C) and a higher recurrence rate (Figure
   [77]1D) than low miR-19a-3p expression was. More information about
   patients is provided in [78]supplementary materials ([79]Supplementary
   Table 2). Collectively, our data suggested that miR-19a-3p might
   function as an oncogenic miRNA and promote HCC progression.

Figure 1.

   [80]Figure 1
   [81]Open in a new tab

   MiR-19a-3p expression in Huh7 cells and HCC tissues. A. Relative
   miR-19a-3p expression in Huh7 cells cultured for 48 h at 1% O[2]
   compared with normoxia (**, p<0.01). B. Relative miR-19a-3p expression
   in HCC tumors and peritumorous tissues (**, p<0.01). C. The correlation
   between miR-19a-3p expression levels in HCC tumors and overall survival
   (p<0.05). D. The correlation between miR-19a-3p expression levels in
   HCC tumors and the recurrence rate among the patients (p<0.05).

MiR-19a-3p promotes HCC growth in vitro and in vivo

   To determine miR-19a-3p function in HCC, its expression level was
   tested in several HCC cell lines. Compared with normal hepatic cells,
   miR-19a-3p was increased in all HCC cell lines ([82]Supplementary
   Figure 1A). Lenti-miR-19a-3pinhibitor was used to silence miR-19a-3p
   expression and the lenti-miR-19a-3p-overexpression vector was also
   constructed. First, miR-19a-3p expression levels were confirmed in the
   overexpression (OE) group and knockdown (KD) group by qRT-PCR (Figure
   [83]2A). The CCK-8 assay results showed that ectopic miR-19a-3p
   expression greatly enhanced Huh7 growth rates, whereas silencing of
   miR-19a-3p significantly inhibited HCC cell proliferation (Figure
   [84]2B), and a similar result was got in Hep3B cell
   line([85]Supplementary Figure 1B). The proliferation-promoting action
   of miR-19a-3p on Huh7 and hep3B were next confirmed in a colony
   formation assay (Figure [86]2C and [87]Supplementary Figure 1C). Cell
   cycle analysis revealed that high miR-19a-3p expression led to a
   greater percentage of cells in the S phase, indicating that the G1-S
   transition was accelerated significantly, whereas miR-19a-3p
   under-expression yielded the opposite phenotype both in Huh7 and Hep3B
   cell lines (Figure [88]2D, [89]Supplementary Figure 1D). Western blot
   analysis showed that cell cycle-related protein cyclin A2 was
   upregulated, and apoptosis-related proteins such as Bad, Bax, and Bak
   were downregulated in the OE group. Contrastingly, the KD group cells
   had the opposite phenotype (Figure [90]2E, [91]Supplementary Figure
   1E). Altogether, the above results suggested that miR-19a-3p induces
   HCC cell proliferation in vitro.

Figure 2.

   [92]Figure 2
   [93]Open in a new tab

   The influence of miR-19a-3p on Huh7 cell growth. A. The effect of
   miR-19a-3p overexpression and inhibition on miR-19a-3p expression
   levels (*, p<0.05). B. The action of miR-19a-3p on Huh7 cell growth
   according to the CCK-8 assay (*, p<0.05). C. The involvement of
   miR-19a-3p in Huh7 cell growth according to a colony formation assay.
   D. Flow cytometry. E. Western blot assay.

   We next studied the influence of miR-19a-3p on HCC growth in vivo. HCC
   cells with low or high miR-19a-3p expression levels were inoculated
   into nude mice. The results showed that the tumors in the mice that
   were injected with miR-19a-3p OE cells were bigger compared with the
   corresponding control, whereas downregulation of miR-19a-3p expression
   significantly repressed the tumor growth (Figure [94]3A). These results
   are consistent with our in vitro data. Ki67 staining indicated that
   miR-19a-3p OE yielded more tumor cells that were Ki67-positive (~80%),
   i.e., more proliferating cells relative to the control were detected.
   There were only ~37% Ki67-positive tumor cells in the control group.
   All these data implied that miR-19a-3p significantly promoted HCC tumor
   growth by improving tumor cell proliferation.

Figure 3.

   [95]Figure 3
   [96]Open in a new tab

   The impact of miR-19a-3p on HCC tumor growth in a xenograft mouse
   model. A. Tumors with different miR-19a-3p expression levels (*,
   P<0.05;**, P<0.01). B. H&E staining C&D. The KI67 assay and statistic
   assay (*, P<0.05;**, P<0.01).

PIK3IP1 mRNA is a direct target of miR-19a-3p

   Generally, miRNAs exert their functions by regulating the expression of
   target genes. Next, to investigate the possible mechanism of action of
   miR-19a-3p, we screened the global gene expression changes between
   miR-19a-3p KD and miR-19a-3p-overexpressing Huh7 cells by
   high-throughout sequencing the mRNA expression profiles. There were 450
   genes that were significantly downregulated, and 431 genes which were
   upregulated ([97]Supplementary Figure 2A). These genes were next
   subjected to Gene Ontology (GO) annotation and pathway enrichment
   analysis. The top 10 GO terms of biological processes were identified,
   and DNA replication and cell cycle checkpoint were the top two items
   (Figure [98]4A). Functional classification of these differentially
   expressed genes based on the Kyoto Encyclopedia of Genes and Genomes
   (KEGG) pathway analysis also demonstrated that these differentially
   expressed genes were highly associated with human T-cell leukemia virus
   infection, the cell cycle, and TNF signaling pathway, among others
   (Figure [99]4B). On the basis of these data, we mainly screened genes
   involved in cell growth and cell cycle as putative target genes of
   miR-19a-3p. RT-PCR was carried out to validate the sequencing results
   (Figure [100]4C).

Figure 4.

   [101]Figure 4
   [102]Open in a new tab

   Screening of numerous genes for miR-19a-3p target genes by a sequencing
   assay, bioinformatics analysis, and a dual luciferase assay. A. GO
   analysis. B. KEGG pathway analysis. C. RT-PCR verification of the
   selected genes. D. The putative target sequence for miR-19a-3p in the
   3′-UTR of PIK3IP1. E. The luciferase assay showed that miR-19a-3p
   inhibited the luciferase significantly (*,p<0.05).

   In combination with the above sequencing analysis, we searched a number
   of databases for putative miR-19a-3p target genes, such as
   microRNA.org, TargetScan, and miRDB, and we found that the 3′-UTR of
   PIK3IP1 contains a complementary site for the seed region of miR-19a-3p
   (Figure [103]4D). To test whether there is a direct interaction between
   miR-19a-3p and the PIK3IP1 gene, we performed a dual-luciferase
   reporter assay using the 3′-UTR of PIK3IP1 and its mutant version
   (contains a mutation in the miR-19a-3p recognition site). Our data
   showed that transient transfection of the wild-type PIK3IP1-luc
   reporter along with miR-19a-3p into Huh7 cells led to a significant
   decrease of luciferase activity as compared with miR-19a-3p empty
   vector control, whereas transient transfection of the mutant
   PIK3IP1-luc reporter together with miR-19a-3p did not yield a
   luciferase activity change (Figure [104]4E). All these observations
   suggested that PIK3IP1 is a direct target of miR-19a-3p in Huh7 cancer
   cells.

MiR-19a-3p regulates cell proliferation via the PIK3IP1-PI3K-AKT signaling
pathway in HCC

   To determine whether miR-19a-3p regulates PIK3IP1 expression in Huh7
   cancer cells, we evaluated the PIK3IP1 expression level in miR-19a-3p
   OE and miR-19a-3p KD groups. The level of PIK3IP1 was markedly reduced
   by ectopic expression of miR-19a-3p, whereas PIK3IP1 expression was
   significantly increased by the KD of miR-19a-3p (Figure [105]5A).
   PIK3IP1 staining of the mouse tumors also showed that elevated
   miR-19a-3p expression led to low expression of PIK3IP1, whereas the KD
   of miR-19a-3p yielded the opposite phenotype ([106]Supplementary Figure
   3A).

Figure 5.

   [107]Figure 5
   [108]Open in a new tab

   The effect of miR-19a-3p on PIK3IP1. A. PIK3IP1 levels in different
   cells ( **, P<0.01). B. The CCK-8 assay for determining the role of
   PIK3IP1 expression in miR-19a-3p OE cells. C A western blot assay of
   expression of relevant proteins in PIK3IP1 OE cells and controls. D.
   The CCK-8 assay for determining the role of PIK3IP1 expression in
   miR-19a-3p KD cells. E. The western blot assay of expression of
   relevant proteins in PIK3IP1 KD cells and controls. F. Schematic model
   of cross talk between miR-19a-3p, PI3K and p-AKT.

   PIK3IP1 is a transmembrane protein that possesses an intracellular
   domain homologous to the p85 regulatory subunit of PI3K; PIK3IP1
   downregulates PI3K activity by binding to a PI3K subunit through a
   specific domain [109]^18. PIK3IP1 is underexpressed in HCC tumors
   compared with the corresponding peritumor tissues ([110]Supplementary
   Figure 3B), consistent with previous studies, which show that PIK3IP1
   suppresses hepatocyte proliferation and HCC progression [111]^19. To
   test whether the dependence of cell proliferation on miR-19a-3p was
   determined by PIK3IP1 downregulation, rescue experiments were
   performed. Huh7 cells with miR-19a-3p OE were transfected with
   lenti-PIK3IP1 or the empty vector. CCK-8 assays revealed that the
   significant proliferation induced by miR-19a-3p was partially reversed
   by introduction of PIK3IP1 (Figure [112]5B), and cell cycle-related
   protein cyclin A2 was downregulated apoptosis-related protein
   expression levels increased (Figure [113]5C). Conversely, the KD of
   PIK3IP1 mimicked the effects of miR-19a-3p and significantly increased
   cellular proliferation (Figure [114]5D); relevant proteins manifested
   the same pattern of expression (Figure [115]5E). AKT and phospho- AKT
   were also quantified to determine the possible mechanism of action of
   the miR-19a-3p-PIK3IP1 axis. We found that cells with a high miR-19a-3p
   level featured low PIK3IP1 expression and an increased p-AKT amount.
   When we restored the PIK3IP1 expression level in these cells, the p-AKT
   amount decreased significantly (Figure [116]5C). This result was
   confirmed by knocking down miR-19a-3p and PIK3IP1 in Huh7 cells (Figure
   [117]5E). All these results indicated that miR-19a-3p promotes cell
   proliferation by downregulating PIK3IP1 and upregulating p-AKT
   expression levels (Figure [118]5F).

Discussion

   MiRNAs are members of the noncoding RNA family and have been reported
   to fine-tune the expression of thousands of mRNAs simultaneously
   through either mRNA degradation or repression of translation. Some
   oncogenic genes such as MYC [119]^20 and some tumor suppressor genes
   such as p53 [120]^21 have been proven as miRNA target genes. Meanwhile,
   miRNA may work as a downstream effector of some important transcription
   factors such as STAT3 [121]^22 and of some long noncoding RNAs
   (lncRNA). Homeostatic imbalance of lncRNAs, miRNAs, and
   cancer-regulatory factors results in biochemical and physiological
   alterations inside the cell, and these are critical parameters for
   cancer progression [122]^23.

   miR-19a is a member of the miR-17-92 miRcluster and the dysregulation
   of the miR-19a is previously found to be associated with multiple types
   of cancer, including breast cancer[123]^24, prostate cancer[124]^25,
   and breast cancer[125]^10. Studies showed that miR-19a inhibitor
   increased p53, Bax and caspase3 expression and decreased the Bcl-2
   expression. miR-19a directly suppressed PPARalpha, promoted cell
   proliferation, tumorigenesis and inflammation by FOXO1 in glioma
   [126]^3^, [127]^9. PTEN is shown as one of target genes, it induced
   cell apoptosis and negatively regulate PI3K/AKT pathway [128]^8.
   miR-19a may also played important oncogenic role in HCC. miR-19a-3p was
   shown to be upregulated in HCC specimens compared with matched tumor
   free tissues, it promoted metastasis and chemoresistiance of HCC via
   repressing PTEN[129]^15. miR-19a overexpression promoted cellular
   proliferation in hep3B and hepG2 cell lines that were neutralized by
   addition of panobinoustat [130]^26. We confirmed miR-19a-3p oncogenic
   role by depressed PIK3IP1 expression, a negative regulator of PI3K.
   However, there are one study showed miR-19a was downregulated in HCC by
   analysis biological data acquisition and integration from literature
   retrieval and inhibit cyclin D1 expression [131]^12. Different type of
   HCC cell lines were used in these studies, and maybe was the reason for
   the discrepancies of those results. The heterogeneity of HCC tissue and
   character of patients may also were reasons, as it is known that miRNAs
   exert specific actions in a context of microenvironment specific
   dependent manner. Additionally, tissue collected, frozen in a short
   time and deposit time were also important because miRNA is easily
   degraded.

   PI3K is a well-known regulator of cell division, motility, and survival
   in most types of cells, and intact PI3K signal transduction is required
   for proper liver function and development. Aberrant PI3K signal
   transduction is implicated in liver tumorigenesis, and PI3K pathway
   constituents are usually altered in human liver cancer. Studies suggest
   that liver cancer features the highest percentage of cases with a
   PIK3CA mutation [132]^27, and another component of the PI3K pathway,
   PTEN, is downregulated in HCC [133]^28. Some studies have shown that
   PTEN is a direct target gene of miR-19a-3p. Here, we found that PIK3IP1
   mRNA is a direct target of miR-19a-3p, and high expression of
   miR-19a-3p caused downregulation of PIK3IP1 in HCC tissues compared
   with the adjacent normal tissues. Those studies indicate that
   miR-19a-3p may be one of the critical elements of the PI3K pathway in
   liver cancer cells.

   MiRNA genes are widespread in all the genomes, and miRNAs may function
   as a prognostic factor. Compared with other cell types, hepatocytes
   have been demonstrated to easily take up oligonucleotides, with the
   implication that miRNA inhibition or replacement might be effective as
   a potential therapeutic approach. Our results support the notion that
   inhibition of miR-19a-3p may be a promising approach for sustaining
   sufficient expression of PIK3IP1 and may be a therapeutic strategy
   against HCC growth.

Supplementary Material

   Supplementary figures.
   [134]Click here for additional data file.^ (1.5MB, pdf)

Acknowledgments