Downregulation of miR‐1225‐5p is pivotal for proliferation, invasion, and migration of HCC cells through NFκB regulation

Abstract Background As one of the most frequently seen malignancies, hepatocellular carcinoma (HCC) serves as the second largest contributor to malignancy‐specific mortality worldwide. MicroRNA‐1225‐5p (miR‐1225) exerts an essential impact on the growth and metastasis of many malignancies. However, the contribution of miR‐125 to HCC and the molecular mechanism of cancer cell viability and apoptosis are still unclear. We focused our research on exploring the function and molecular mechanism of miR‐1225 in regulating HCC cell growth, migration, and invasion. Material Quantitative PCR data showed that miR‐1225 expression was repressed in HCC cell lines and in the tissues of HCC patients, compared to that in normal human hepatic cells and tissues. Transfection of a miR‐1225 mimic inhibited cell viability and proliferation as indicated by CCK‐8 staining and MTT assay. Transwell invasion, wound healing assay, and Western blotting were performed to assess whether miR‐1225 repressed the metastasis and invasion of HCC cells, and decreased matrix metalloproteinase 9 (MMP9) expression. Further bioinformatic prediction and dual‐luciferase reporter assay suggested that miR‐1225 targeted the 3′‐UTR of NFκB p65. Results Overexpression of p65 protein counteracted the repressive impact of miR‐1225 on invasion, migration, and proliferation of HCC cells. Conclusion This research provided new evidences that miR‐1225 inhibits the viability, migration, and invasion of HCC cells by downregulation of p65.


| INTRODUC TI ON
Hepatocellular carcinoma (HCC) serves as one of the ten most prevalent malignancies worldwide with respect to incidence and mortality. 1 It is characterized to have a bad clinical outcome and shows metastasis to distant regions. 2,3 Despite emerging studies investigating migration and invasion, metastasis is still the dominant contributor to HCCspecific mortality as in other malignancies. [4][5][6][7][8] Even though its diagnostic process and treatment have advanced substantially, its mortality rate has remained considerably high in the last 5 years. Therefore, there is an imperative need to investigate and understand the innate mechanism that dictates metastatic behavior in HCC cells.
According to clinical records, the high invasive capability of malignant cells is related to poor survival, suggesting the aggravating contribution of strong invasive capability to the malignant development of cancer. 9,10 In HCC, macrophagocytes observed inside primary neoplasms are a general indicator of tumor progression and metastasis. [11][12][13][14] Macrophagocytes, after activation, are critical for the development and invasive process of tumors as they selectively upregulate matrix-metalloproteinases (MMPs), which disrupt the extracellular matrix and undermine the basement membrane. 15 As small single-stranded RNAs without coding functions, miRs contain eighteen to twenty-two nucleotides and are endogenously expressed in multiple organisms ranging from animals to plants. 16,17 Various studies have revealed the essential impact of miRs on different biological reactions including apoptosis and migration in malignancy. Few studies have revealed the tumor repressive impact of miRNAs in HCC. 18,19 It is previously reported that miR-1225 expression is repressed in human gastric carcinoma and inhibits metastasis and proliferation of malignant cells. 20 Aberrant excessive miR-1225 expression repressed migration, proliferation, and invasion of glioblastoma cells in vitro. Furthermore, excessive miR-1225 expression impaired the growth of glioblastoma xenograft malignancy by targeting insulin receptor substrate 1 (IRS1). 21 Additionally, miR-1225 serves as a malignancy repressor in laryngeal and pancreatic malignant cells by targeting CDC14B and JAK1. 22,23 This study examines the impact of miR-1225 on HCC cells.
Repressed miR-1225 expression was revealed in HCC tissue specimens and in HCC cells. Mechanistic exploration proved that miR-1225 inhibited the migration, invasion, and proliferation of HCC cells in vitro.
Furthermore, miR-1225 was found to directly target NFκB p65 in HCC.

| Clinical samples
The study group included ten cases of HCC, with the age of the subjects ranging from 30-60 years. Clinicopathological characteristics were acquired from medical records. We harvested the para-neoplastic specimens at no less than 1 cm from the malignant specimens. All cases were hepatectomy candidates from Jining No.1 People's Hospital.
The study was approved by the Ethical Committee of the Jining No.1 People's Hospital. Fully informed consent was acquired from each patient and doctor with regard to specimen usage. Diagnosis and review were performed independently by three pathologists.

| Cells and transfection
Human Huh-7 and HepG2 cells were acquired from the Cell Bank of Academia Sinica (Shanghai, China), and were cultured in DMEM medium with 10% FBS. Cells were maintained at 37°C with 5% CO 2 .

| MiR-1225 mimic/prohibitor preparation
The miR-1225 mimic and negative control (NC) were obtained from RiboBio. NC and the miR-1225 mimic were added to 0.9% NaCl to a final concentration of 10 mg/mL.

| MTT Assessment
MTT assay was performed to assess cell proliferation. Briefly, cells were supplemented with twenty microliters of MTT (0.5 mg/mL) and incubated for four hours at 37°C. The supernatant was discarded, and 150 µL of DMSO was added to every well and rotated for ten minutes to dissolve the purple formazan crystals formed. Absorbance was measured on an Infinite M200 microplate reader (Tecan) at 490 nm.

| EdU proliferation test
The cells were washed three times with PBS and then stained with 300 µL of EdU staining solution for 2 hours according to the manufacturer's protocol. After an additional three washes with PBS, the cells were examined with a fluorescence microscope (Olympus).
Proliferating cells were identified by red staining, and the number of proliferating cells in six different fields was counted.

| RNA extraction and quantitative PCR
Total RNA was separated from cells using TRIzol reagent. For, qPCR, the reaction mixture included cDNA, forward and reverse primers, as well as SYBR Green PCR Master Mix in a total volume of 20 µL and was amplified in the Light-Cycler 480 Real-Time PCR system (Roche, Basel, Switzerland). GAPDH served as the internal reference. Quantification was carried out using the 2 −ΔΔCT approach via normalization against GAPDH.

| Wound healing test
Cells (1 × 10 5 cells/mL) were seeded in 24-well plated after 48 h of treatment and were incubated until 70%-80% confluency. Using a sterile pipette tip, a scratch wound was created by scraping the cell monolayer. Fresh medium was added, and the cells were incubated for further 48 hours or were not incubated (control). Cells were imaged under an inverted microscope (Nikon).

| Dual-luciferase reporter test
miR-1225 was predicted to target the 3′-UTR of p65. Thus, a wild type or mutated sequence of the predicted target site was generated. Dual-Luciferase Reporter Assay system was use to examine luciferase function at 48 hours subsequent to transfection as instructed (Promega). Renilla luciferase function was used to normalize the firefly luciferase activity.

| Statistical analysis
Outcomes were displayed as mean ± SD. One-way ANOVA or Student's t tests were applied to evaluate the differences. P < .05 was considered significant.

| MiR-1225 expression is decreased in HCC cells and in tissue specimens from HCC patients
Expression of microRNAs is relevant for the prognosis and diagnosis of different cancers. Our research revealed that miR-1225 was remarkably downregulated in HCC samples compared to that in normal healthy samples ( Figure 1A). miR-1225 concentration in HepG2 and in Huh-7 cells was compared with that in normal human hepatic

| MiR-1225 repressed the proliferation of HCC cells
To confirm that miR-1225 regulates survival and proliferation of HCC cells, Huh-7 and HepG2 cells were transfected with a miR-1225 mimic or NC mimic (Figure 2A, 2B). MTT assay showed that proliferation of Huh-7 and HepG2 cells was reduced subsequent to transfection by the miR-1225 mimic at 12-72 hours post-transfection ( Figure 2C, 2D). miR-1225 upregulation caused a noticeable decrease in CCK-8-positive cell numbers, but transfection with the NC mimic did not influence cell proliferation assessed by CCK-8 ( Figure 2E, 2F). In the wound healing assay, miR-1225 inhibited the migration of Huh-7 and HepG2 cells toward the gap created by scratching the cell monolayer ( Figure 3C, 3D), which was consistent with the outcome of the Transwell assay. Our study also explored the contribution of miR-1225 to MMP-9 expression, which is critical for migration and invasion capabilities of malignant cells. Ectopic upregulation of miR-1225 led to a decrease in MMP-9 in both cell lines, as determined by Western blotting (Figure 3E, 3F). These data suggest that miR-1225 represses the invasive and migratory capacities of HCC cells in vitro.

Luciferase activity was repressed by eighty-five percent in cells that
were transfected with the miR-1225 mimic fused to the p65 3′-UTR compared to the control group.
MiR-1225 expression was subsequently assessed in HCC cells and in tissues. HCC displayed p65 upregulation compared to that in normal liver tissues ( Figure 4C). NFκB p65 concentration in HepG2 and Huh-7 cells was elevated compared with that in normal liver cells ( Figure 4D, 4E). Western blotting and qPCR were utilized to assess the contribution of miR-1225 mimic to p65 expression in HCC cells.
Transcription and translation of p65 were reinforced subsequent to transfection with the miR-1225 mimic ( Figure 4F-4I). It was thus proved that p65 was downregulated subsequent to miR-1225 silencing and that miR-1225 targeted the p65 3′-UTR.

| P65 overexpression counteracts the repressive impact of miR-1225 on the proliferation, migration, and invasion of HCC cells
To assess whether p65 contributes to the repressive impact of miR-1225 on the properties of HCC cells, p65 was overexpressed F I G U R E 3 miR-1225 upregulation inhibited HCC cell migration and invasion. Subsequent to transfection with the miR-1225 mimic or NC mimic, (A, B) invasion and (C, D) migration of HepG2 and Huh-7 cells was examined using the Transwell migration test and wound healing assay. (E, F) Western blotting was performed to determine the MMP-9 expression in HCC cells expressing miR-1225 F I G U R E 4 miR-1225 targets p65. A, Graphical representation of the conserved miR-1225 binding motif at the 3′-UTR of NFκB p65. B, Luciferase function exhibited via luciferase reporter constructs carrying either the wild type (WT) or mutated (MU) human p65 3′-UTR subsequent to miR-1225 mimic transfection. Luciferase function was normalized to the function of Renilla luciferase. C, p65 expression in samples acquired from HCC patients (n = 10) vs. normal healthy tissue (n = 10) was examined by qPCR. D, Western blotting and E, qPCR were carried out in order to examine p65 expression in Huh-7, HepG2, and normal liver cells. F, G, Western blotting and H, I, qPCR were conducted to assess p65 protein expression and transcription in HCC cells, respectively, subsequent to transfection with the miR-1225 mimic and NC mimic. Outcome is displayed as mean ± SD. *P < .05, ***P < .001 vs control group F I G U R E 5 NFκB p65 is involved in miR-1225-inhibited proliferation, invasion, and migration of HepG2 and Huh-7 cells. A, B, HepG2 and Huh-7 cells underwent co-transfection with of pCDNA3-p65/ pCDNA3 and the miR-1225 mimic to upregulate the p65 expression. A, C, qPCR and B, D, WB were performed to detect the p65 protein expression in both HCC cell lines. E, F, Proliferation of Huh-7 and HepG2 cells was measured at 12, 24, 36, 48, 60, and 72 hours after transfection, using the MTT assay. G, H, Invasion and I, J, migration of HepG2 and Huh-7 cells was examined using the Transwell migration test and wound healing assay. Outcome is displayed as mean ± SD. *P < .05 vs control group in two HCC cell lines, that also underwent transfection with the miR-1225 mimic. Western blotting and qPCR were used to confirm p65 upregulation in both HCC cell lines ( Figure 5A-5D). p65 overexpression led to the proliferation recovery of those two cell lines repressed by miR-1225, as evidenced by the MTT assay ( Figure 5E, 5F). Upregulation of p65 resulted in a remarkable increase in the quantity of invasive HCC cells subsequent to transfection, using the Transwell assay ( Figure 5G, 5H). Further, p65 remarkably increased the quantity of migrated Huh-7 and HepG2 cells, as determined by the wound healing assay ( Figure 5I, 5J). Thus, p65 shows a protective role on the migration and proliferation of HCC cells. Nuclear factor kappa B (NFκB) is a critical survival factor in various physiological reactions, such as embryonic liver development and liver regeneration. 25 In liver cancers, NFκB is a dominant contributor to the resistance to TNF cytotoxicity and to functional pathways such as TNF receptor-associated factor 2. 26 NFκB serves as an essential regulator in the suppression of cell death reactions, and NFκB stimulation has been revealed to elevate cell death counteracting threshold of cells and tissues exposed to cytotoxic cytokines including TNF by repressing the triggering of caspase-8 stimulation. 27 On the contrary, NFκB exerts various wide-ranging impacts regulated via a complicated modulating network of repressors and coactivators. 28 Proteases like MMP-9 are required for the invasion and destruction of extracellular matrix, thus boosting the invasive progression of cancerous cells to nearby healthy tissues. 29-31 MMP-9 from tumor and stromal cells, particularly macrophagocytes, has an indispensable position in the invasive, migratory, and angiogenic progression of malignant tumors. As NFκB is the key transcription factor for MMP9 expression, 32 we hypothesize that NFκB could participate in regulating cancer cell metastasis by mediating MMP-9 expression. The current study showed that downregulated miR-1225 or increased NFκB expression in HCC samples was related to increased proliferation, migration, invasion, and enhanced MMP-9 expression. Our results demonstrate that miR-1225, to some extent, could downregulate NFκB-induced MMP-9 elevation in HepG2 and Huh-7 cells.

| D ISCUSS I ON
In conclusion, this research indicates a malignancy counteracting impact of miR-1225 on 2 HCC cell lines and suggest that HCC progression involves NFκB. However, we are still unable to rule out other potential factors that could be targeted by miR-1225 and thus lead to HCC progression. Thus, additional cross talk assays are needed in future to explore and recognize the targets for biotin-labeled miR-1225 in HCC tissue specimens from patients and cell lines. This approach could offer a more accurate and profound interpretation for the contribution of miR-1225 to HCC generation.