miR‐1249‐3p accelerates the malignancy phenotype of hepatocellular carcinoma by directly targeting HNRNPK

Abstract Background microRNAs (miRNAs) have been implicated to play crucial roles in carcinogenesis. miR‐1249‐3p was reported to be abnormally expressed in multiple human cancers. However, its biological role and the associated underlying mechanisms in hepatocellular carcinoma (HCC) remain largely unknown. Methods miR‐1249‐3p expression level in HCC cell lines and normal cell line was measured by quantitative real‐time PCR. Role of miR‐1249‐3p on HCC cell proliferation, colony formation, and invasion was examined by cell counting kit‐8 assay, colony formation assay, and transwell invasion assay, respectively. Luciferase activity reporter assay and western blot were performed to validate whether heterogeneous nuclear ribonucleoprotein K (HNRNPK) was a direct target of miR‐1249‐3p. Effect of miR‐1249‐3p on overall survival of HCC patients was analyzed at KM Plotter website. Results We found miR‐1249‐3p expression level was increased, while HNRNPK expression level was decreased in HCC cell lines compared with normal cell line. Knockdown miR‐1249‐3p expression inhibits HCC cell proliferation, colony formation, and cell invasion through regulating HNRNPK in vitro. We also showed high miR‐1249‐3p expression was a predictor for poor overall survival of HCC patients. Conclusions These findings about miR‐1249‐3p/HNRNPK pair provide a novel therapeutic method for HCC patients.


| INTRODUCTION
Hepatocellular carcinoma (HCC) is a major health threat worldwide (Bray et al., 2018). Most of HCC cases were found in advance stages as it is difficult for early diagnosis (Ghouri, Mian, & Rowe, 2017). Emerging evidence has indicated that HCC tumorigenesis is associated with dysexpression of multiple key genes and signaling pathways (Yin et al., 2016;Zhou, Du, Kong, Zhang, & Chen, 2018). Thus, it is imperative to investigate molecular mechanisms behind HCC tumorigenesis to explore novel biomarkers for cancer diagnosis or treatment. microRNAs (miRNAs) are reported to regulate majority of human genes, and hence to play a crucial role in cancer initiation and progression, including HCC (Krol, Loedige, & Filipowicz, 2010). For example,  expression level was decreased in HCC tissues compared with normal tissues (Babu & Muckenthaler, 2019). Overexpression of miR-148a using miR-148a mimic significantly decreased HCC cell proliferation through targeting transferrin receptor 1 (616740) (Babu & Muckenthaler, 2019).  expression was downregulated in HCC tissues and cell lines in comparison with normal tissues and cell lines (Wu et al., 2019). Force miR-29c-3p expression inhibited HCC progression in vitro and in vivo through regulating large tumor suppressor gene 1 (603473) (Wu et al., 2019).  was downregulated in HCC and correlated with lymph node metastasis and TNM stage . Furthermore, they should overexpression of miR-195 could promote HCC cell malignancy behaviors, while the knockdown of miR-195 will cause the opposite effects.
miR-1249 was previously reported to be abnormally expressed in several human cancers Fang, Li, Xu, Hui, & Li, 2018;Ye et al., 2017). For instance, miR-1249 was found upregulated expression in glioma tissues and cell lines, and overexpression of miR-1249 enhanced glioma progression in vitro and in vivo through targeting adenomatous polyposis coli 2 (Fang et al., 2018). In colorectal cancer, miR-1249 was reported as a transcriptional target of P53 (191170) to suppress tumor growth, metastasis, and angiogenesis . Importantly, miR-1249 expression was found could activated by Hedegehog signaling pathway to stimulate HCC progression (Ye et al., 2017). Nevertheless, the biological role and molecular mechanisms of miR-1249-3p in HCC remain to be elucidated.
In current study, we measured miR-1249-3p expression level in HCC cell lines and normal cell line. HCC cell proliferation, colony formation, and invasion after synthetic miRNAs transfection were examined by cell counting kit-8 assay, colony formation assay, and transwell invasion assay, respectively. Luciferase activity reporter assay and western blot assay were conducted to validate heterogeneous nuclear ribonucleoprotein K (600712, HNRNPK) as a direct target of miR-1249-3p. Effect of miR-1249-3p expression on overall survival of HCC patients was analyzed using bioinformatic tool.

| Cell line and cell culture
HCC cell lines (Huh7 and Hep3B) and normal liver cell line L02 obtained from Cell Bank of Chinese Academy of Sciences (Shanghai, China) were incubated at Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Inc.) and 1% penicillin/ streptomycin (Thermo Fisher Scientific, Inc.) at a 37°C humidified incubator containing 5% CO 2 .

| Cell transfection
miRNA inhibitor (miR-inhibitor) and the corresponding negative control (miR-NC) were purchased from GenePharm. Small interfering RNA targeting HNRNPK (siR-HNRNPK) and the negative control (siR-NC) were also obtained from GenePharm. Cell transfection was conducted using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.

| Cell counting kit-8 (CCK-8) assay
Cells were seeded into 96-well plate with the density of 5 × 10 4 cells/well. 0, 24, 48, and 72 hr after seeding, CCK-8 reagent (Beyotime) was added to each well and further incubated for 4 hr. Optical density at 450 nm was recorded to assess cell proliferation.

| Colony formation assay
Cells were plated in six-well plates at the density of 500 cells/ well and incubated for 2 weeks. Colonies formed were fixed with methanol and stained with crystal violet. Colonies number was counted from at least five independent fields under microscope.

| Kaplan-Meier curve analysis
Kaplan-Meier plotter (www.kmplot.com, Nagy, Lánczky, Menyhárt, & Győrffy, 2018) was employed to analyze miR-1249-3p expression on overall survival of HCC patients. Cut-off value was auto selected in the algorithm. Log-rank test was used to analyze difference in groups.

| Statistical analysis
Data were analyzed at SPSS 19.0 (SPSS Inc.) and then presented as mean ± SD. Statistical analysis was analyzed using two-tailed Student's t test or one-way ANOVA and Tukey's post hoc test. Differences were considered as statistically significant when p < .05.

| Upregulation of miR-1249-3p in HCC
qRT-PCR revealed mature miR-1249-3p expression level was significantly increased in HCC cell lines (Huh7 and Hep3B) compared with normal liver cell line L02 ( 1a). Moreover, we found high miR-1249-3p expression was a predictor for poor overall survival of HCC patients (Figure 1b).

| miR-1249-3p regulates HCC cell behaviors via targeting HNRNPK
We then investigate whether HNRNPK was a functional target of miR-1249-3p by rescue experiments. Introduction of si-HNRNPK significantly decreased HNRNPK expression level in HCC cells (Figure 4a). CCK-8 assay and colony formation assay indicated cell growth was increased by si-HNRNPK (Figure 4b,c). Meanwhile, we showed cell invasion ability was also increased by si-HNRNPK ( Figure 4d). Importantly, we found N-cadherin and Vimentin expression was increased, while E-cadherin expression was decreased by si-HNRNPK ( Figure 4e). Moreover, the effect of miRinhibitor on cell growth and invasion ability could partially reversed by si-HNRNPK (Figure 4b-e).

| DISCUSSION
In previous study, miR-1249 expression was reported could be activated by Hedegehog signaling pathway in HCC (Ye et al., 2017). However, the downstream targets of miR-1249 are largely unknown. In this study, we showed miR-1249-3p expression was significantly increased in HCC cell lines compared with normal cell line L02. Moreover, we showed high miR-1249-3p expression was a predictor for poor overall survival of HCC patients, indicating miR-1249-3p may function as an oncogene in HCC. Loss-offunction experiments showed knockdown of miR-1249-3p inhibited HCC cell proliferation, colony formation, and invasion in vitro. Importantly, we showed several key players in epithelial-mesenchymal transition (EMT) process could be regulated by miR-1249-3p. EMT is a process to transfer epithelial cell into mesenchymal phenotype, an indicator for malignancy behaviors of human cell (Tania, Khan, & Fu, 2014).
As small regulator molecules, miRNA regulates target gene expression at posttranscriptional level (Iqbal, Arora, Prakasam, Calin, & Syed, 2018). It has been widely recognized that one gene can be regulated by multiple miR-NAs and one miRNA could regulate various genes in a cell context dependent manner (Iqbal et al., 2018). Previous studies identified several targets of miR-1249 in human cancers Fang et al., 2018). Here, we found HNRNPK was a potential target of miR-1249-3p. HNRNPK is a crucial RNA and DNA binding protein and reported function as a crucial regulator for the progression of human cancers (Barboro, Ferrari, & Balbi, 2014). HNRNPK is involved in multiple biological processes including chromosome remodeling, DNA transcription, RNA processing, and RNA translation (Bomsztyk, Denisenko, & Ostrowski, 2004;Mikula et al., 2006). HNRNPK overexpression can retard gastric cancer cell proliferation and colony formation in vitro and in vivo through regulating p53/p21/CCND1 axis, indicating a tumor suppressive role of HNRNPK . On the contrary, HNRNPK overexpression was found could increase pancreatic cancer cell proliferation, migration, and invasive (He et al., 2017). Here, we validated HNRNPK was a direct target of miR-1249-3p using luciferase activity reporter assay and western blot assay. Moreover, rescue experiments demonstrated that HNRNPK was a functional target of miR-1249-3p.
In summary, miR-1249-3p/HNRNPK axis could affect EMT, proliferation, colony formation and invasion of HCC cells. We therefore hypothesized that miR-1249-3p inhibitor might be a therapeutic agent for HCC but further in-depth investigations are required.