miR199a represses the proliferation of hepatocellular carcinoma cells by targeting SGK3 through Akt/mTOR pathway

doi:10.21203/rs.3.rs-3754567/v1

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miR199a represses the proliferation of hepatocellular carcinoma cells by targeting SGK3 through Akt/mTOR pathway

https://doi.org/10.21203/rs.3.rs-3754567/v1

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Purpose

Previous studies have suggested that some microRNAs (miRNAs) play a vital role in carcinogenesis and progression of hepatocellular carcinoma (HCC). As one of these newly found miRNAs, miR-199a-3p (miR199a), has been shown to be highly involved in the regulation of HCC growth. However, the mechanism underlying miR199a regulation in HCC remains unclear.

Methods

In this study, the evaluation of miR199a expression in HCC cells (HCCs) and tissues was evaluated by RT-qPCR. Bioinformatics analysis and luciferase reporter assay were performed to prove the regulation effect of miR199a on SGK3. CCK-8 assay was performed to assess the proliferation of HCCs (HepG2 and HuH7) after transfection. Subsequently, the biological function of miR199a/SGK3 axis in HCCs was investigated.

Results

Herein, we found that miR199a is usually underexpressed in the HCC tissue and cell lines (HepG2 and HuH7) compared with their corresponding control. The expression pattern of SGK3, as a direct target of miR199a, was opposite that of miR199a. shRNA-mediated silencing of SGK3 decreased the proliferation of HCC cells (HCCs) in vitro; however, these phenotypes were promoted by miR199a mimics. Mechanistically, miR199a inhibited the proliferation of HCCs through the downregulation Akt/mTOR signalling by targeting SGK3.

Conclusion

our findings demonstrated an important role for SGK3 as a miR199a target and a mediator of the Akt/mTOR signaling pathway. These findings highlight the miR199a/SGK3 axis as a potential therapeutic target for managing HCC.

hepatocellular carcinoma

miR199a

SGK3

mTOR

cell proliferation

Hepatocellular carcinoma (HCC) is a primary liver tumour with a high mortality rate, it has become the third greatest cause of cancer deaths worldwide(905,677 in 2020 )(Sung et al. 2021). According to a 2022 study, liver cancer was the second most common cancer in China, there were 336,400 liver cancer deaths in 2016(Li et al. 2022). Hepatitis virus infections, excessive alcohol consumption and nonalcoholic steatohepatitis have been identified as risk factors for HCC. Despite recent advances in surgical techniques and perioperative management, the prognosis of HCC patients after hepatectomy remains unsatisfactory due to the high recurrence rates after surgical resection(Vitale et al. 2023). Thus, there is an urgent need for novel therapies and chemotherapeutic drugs that target critical molecules required for tumour growth of HCC. Although there are many studies which have been conducted on the molecular mechanism of HCC, the crucial molecular mechanisms underlying the development of HCC remain largely unknown, hindering the development of new pharmacological therapies. Therefore, elucidating the molecular mechanism of HCC and identifying treatment objectives are urgently needed and necessary for the development of HCC therapeutic strategies.

MicroRNAs (miRNAs) are endogenous short non-coding RNA containing 21–24 nucleotides, which are widely found in mammalian cells and play a major role in various biological phenomena, such as cell differentiation, immune responses, tumourigenesis, and so on. The mature miRNAs are mostly distributed in the cytoplasm and mainly function as mRNA suppressors through binding on the complementary sequences of 3’-untranslated regions (3'UTR)(Anijs et al. 2023). Thus, a miRNA can regulate multiple mRNAs, and involved in many physiological and pathological processes(Mehterov. 2023). The previous studies have reported that miRNA plays a critical role in HCC progression through regulating cell proliferation, apoptosis, migration and metabolism(Shen et al. 2021). It has been demonstrated that miR-10b, miR-221, miR-21 and miR-1269b promote the proliferation of HCCs, whereas miR-193a-5p, miR-449, miR-501-3p and MR-541 suppress HCC cell proliferation and metastasis(Chen et al. 2020; Komoll et al. 2021; Liu et al. 2021; Luo et al. 2018; Roy et al. 2018; Sandbothe et al. 2017; Tian et al. 2019; Xu et al. 2020; Yuan et al. 2013). Although a series of miRNAs have been identified underlying the molecular mechanism of these miRNAs in HCC, they still remain to be explored.

In the present study, we found that the miR199a was down-regulated in HCC with greatest significance via analyzing miRNA microarray data from liver tumour tissue and normal liver tissues, thus, we supposed the miR199a had played a key role in HCC. however, in view of the functional characteristics, the crucial direct target of miR199a has not yet been revealed. Subsequently, we utilised in vitro assays to demonstrate that miR-199a repressed the proliferation of HCCs by directly suppressing serum and glucocorticoid-regulated kinase 3 (SGK3), which concomitantly modulated the Akt/mTOR signalling pathways. We provide evidence that targeting miR199a/SGK3 signalling may be a feasible rationale to develop meaningful therapeutic strategies against HCC.

Acquisition of microarray data

The HCC and control tissue microarray data was download from Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) database, which was submitted by Morita K (Series Accession: GSE41874, ID:200041874).

Patient samples

Control and tumour tissues were obtained after biopsy in the pathology department of Xiangyang No.1 People's Hospital (Xiangyang, China). All the tissues were from male patients (39 to 60 years old). This work was reviewed and acquired the approval of the Ethics Committee of Xiangyang No.1 People's Hospital(XYYYE20230121), and all the patients had written informed consent.

Cell culture and miRNA transfection

The HepG2, HuH7 and 293T cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The normal human hepatocyte L02 cell line was purchased from the China Center for Type Culture Collection (Shanghai, China). Cells were cultured in DMEM (Invitrogen, Carlsbad, USA) with 10% FBS (Invitrogen) under 5% CO2 at 37°C.

The miR199a mimic and negative control were synthesized by RiboBio (Guangzhou, China). miR199a-mimics (mimics): 5’-ACAGTAGTCTGCACATTGGTTA-3’, and the negative control mimics (NC-mimics) sequence was 5’-GTGTTTGATGACCCGTTTA-3’. Cells (5 × 10⁵ cells per well) were seeded in a 6 well plate. When the cells were at 85% confluence, the mimics or NC-mimics were transfected into cells at a final concentration 50 nM using Lipofectamine™ 2000 Transfection Reagent (cat.no.11668019, Invitrogen, Carlsbad, USA). Briefly, dilute 2 amounts of Lipofectamine™ Stem Reagent and miRNA mimics in Opti-MEM™ I Medium, respectively. Next, mixed the diluted miRNA mimics and diluted Lipofectamine™ Stem Reagent (1:1 ratio), incubated for 20 min at room temperature. Lastly, add miRNA mimics-lipid complex to cells.

miRNA target prediction

The miR199a directly targeting genes were predicted on the bioinformatics prediction websites TargetscanHuman 7.1 (http://www.targetscan.org/), and the whole predicted targets data was downloaded for analysing.

Cell Counting Kit-8 (CCK-8) assay

HepG2, HuH7 cells were seeded in 96 well plates (~ 2000 cells per well) with 100 µl culture medium for 0 h, 24 h, 48 h and 72 h, respectively, after which CCK-8 (Dojindo, Japan) was added to wells (10 ul per well) and incubated (37 ℃, 2 h), then, we recorded the absorbance of each well at 450 nm and analysed the relative cell number to assess cell proliferation.

5-Ethynyl-2'-deoxyuridine (EdU) incorporation assay

HepG2 and HuH7 cells were transfected with mimics or NC-mimics for 24 h in 96 well plates, amd the cell proliferation was tested by Cell-Light EdU Apollo567 In Vitro Kit (cat.no.C10310-1, RiboBio, Guangzhou, China) according to the manufacturer’s protocol¹. Briefly, post miRNA mimics transfected 24 h, cells were incubated with 50µM EdU for 2 h at 37℃ and fixed with 4% paraformaldehyde for 30 min at room temperature. Next, incubated with 2 mg/mL glycine for 5 min, after that, cells were incubating 0.5% TritonX-100 for 10 min. Thirdly, cells were incubated with 1X Apollo® for 30 min avoid light at room temperature. Lastly, the 1X Hoechst 33342 was used for DNA staining. Olympus FV500-IX71 microscope (Olympus, Tokyo, Japan) was used to acquire images.

GO and KEGG analysis

To uncover the crucial target of miR199a, the 469 predicted target genes of miR199a were performed by GO term and KEGG pathway enrichment analysis on DAVID Bioinformatics Resources 6.8 (https://david.ncifcrf.gov/). The enrichment index with P < 0.05 was considered to be statistically significant.

Stable SGK3 knockdown cell line construction

To construct and filtrate stable SGK3 knockdown cell lines, we used the lentivirus with puromycin resistance. The pLKO.1-shSGK3 or pLKO.1‐shRNA (negative control) plasmid andpsPA×2 and pMD2.G were co-transfected into HEK293T cells using lipofectamine® 2000 for incubating for 20 min at room temperature, then the culture medium was collected after 48 h and added to HepG2 and HuH7 cells with polybrene in a 10 µg/mL concentration (H9268; Sigma, St. Louis, MO, USA), and incubated for 24 h. Next, the cells were filtrated through 2 µg/mL puromycin (A1113803; Gibco, Grand Island, NY, USA). The lentivirus infected cells were puromycin-resistant, whereas the uninfected cells were not, therefore, the uninfected cells were removed by puromycin. Finally, the stable knockout cells were cultured and amplified for subsequent experiments. The shSGK3 sequence, 5’-CCGGGCCGAGATGTTGCTGAAATGTCTCGAGACATTTCAGCAACATCTCGGCTTTTTG-3’; the sequence for the shRNA: 5’-CCGGCCTGACCCTGAAGTTCATCTGCACGTGCAGATGAACTTCAGGGTCATTTTTG-3’.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA from frozen patient samples and cell lines was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA was converted to cDNA using the FastQuant RT kit (TIANGEN Biotech, Beijing, China) or Bulge-Loop miRNA qRT-PCR Starter Kit (Ribobio, Guangzhou, China). RT-qPCR was then conducted using SYBR Green PCR Master Mix (Invitrogen, Carlsbad, CA, USA). U6 small RNA and β-actin were used as endogenous control of miRNA and mRNA, respectively. The primers for miR199a and U6 were obtained from RiboBio (Guangzhou, China). The primer sequences used were designed and synthesised by Sango Biotech, Shanghai, China (Table S1). All samples were run in triplicate. The data analyses were performed with SDS 2.2.2 software.

Western blot assay

Proteins were electrophoresed by 10% SDS-PAGE and transferred to PVDF membranes (cat. no. AR0136-04, BOSTER, Wuhan, China). Next, the membranes were blocked in non-fat dry milk (~ 5%) for 40 min at room temperature on a shaker and incubated with special primary antibodies (1:1000) at 4°C overnight. Further, the membranes were washed and incubated with peroxidase-conjugated secondary antibody (anti-rabbit) in the blocking buffer for 1 h. Protein expression levels were visualised using an enhanced chemiluminescence kit (cat. no. P0020; Beyotime Institute of Biotechnology, Haimen, China). ImageJ software (National Institutes of Health Software) was used for Western blot densitometric analysis. Primary antibodies: SGK3 antibody, Cell Signalling Technology, (USA; cat. no. 8573S, dilution 1:1000); ACTIN antibody, BOSTER, (China; cat. no. BM0005, dilution 1:1000); AKT antibody, Cell Signalling Technology, (USA; cat. no. 9272, dilution 1:1000); Phospho-AKT^{Ser 473}(p-AKT) antibody, Cell Signalling Technology, (USA; cat. no. 4060, dilution 1:1000); mTOR antibody, Cell Signalling Technology, (USA; cat. no. 2972, dilution 1:1000); Phospho- mTOR^Ser2448 (p-mTOR) antibody, Cell Signalling Technology, (USA; cat. no. 2971, dilution 1:1000); PCNA antibody, ThermoFisher Scientific, (USA; cat. no. 13-3900, dilution 1:1000).

Luciferase reporter assay

The 3ʹ-UTR or 3ʹ-UTR mutated sequence of SGK3 was synthesised and inserted into into the pmirGLO dualluciferase reporter plasmids (Promega, Madison, WI, USA). HEK293 cells overexpressing miR199a or its control were transfected with SGK3-3ʹ-UTR-Wt and SGK3-3ʹ-UTR-Mut. Post transfection for 24h, cells were lysed and extracts were prepared for luciferase activity detection with the kit (Promega, Madison, WI, USA).

Statistical analyses

Data are expressed as the means ± standard error of the mean (SEM) from at least three independent assays, analysed by GraphPad Prism version 7.0 (GraphPad Software Inc., La Jolla, CA, USA). Statistical differences were analysed by Student’s t-test. P < 0.05 was considered to be statistically significant and achieved significant enrichment.

miR199a was down-regulated in HCC tissue and cells lines

Via analysing the miRNA expression profile data that was downloaded from GEO database, the differential expression of miRNAs in HCC was obtained (Fig. 1a). Among these miRNAs with differential expression, the miR199a has the highest down-regulated multiples. Further, the relative expression of miR199a in the HCC tissue was detected, and the results reflected that miR199a showed a 3-fold down-regulation in the HCC tissue compared to adjacent no-tumour tissues (Fig. 1b). Kaplan–Meier survival analysis revealed that miR199a with low expression in HCC tissue and it exhibited shorter survival probability compared with these which was high expression.Although no meaningful p value (P = 0.086) was shown, the trend showed that the expression level of miR199a was positively correlated with the survival probability (Supplementary Figure S1). In order to find a suitable cell lines for the following study, the relative expression of miR199a in the normal liver cell line (L02) and HCC cell lines (HepG2 and HuH7) were detected. Similarly, compared to the L02, the expression level of miR199a in HepG2 and HuH7 showed a significant decrease (Fig. 1c).

Overexpression of miR199a suppressed the proliferation of HCCs

To further investigate miR199a function on HCC, the miR199a mimics was transfected into HepG2 and Huh7 cells. As depicted in Fig. 1d & 1e, miR199a levels were significantly increased in miR199a mimics group but not in lipo and NC-mimics groups. The results of the CCK-8 assay indicated that overexpression of miR199a significantly inhibited the proliferation of HepG2 and HuH7 cells at the 72 h time point, compared with the mimic control group (Fig. 1f & 1g). Moreover, HepG2 and HuH7 cells treated with miR199a mimics resulted in a significant decrease of the EdU uptake, leading to a deficiency of EdU positive cells compared with untreated cells (Fig. 1h & 1i). These findings indicated that miR199a act as a potential tumour suppressor in HCC.

PI3K/Akt signalling pathway was the miR199a target pathway

In order to understand possible mechanisms in which miR199a inhibits the proliferation of HCCs, 469 potential target genes of miR199a were predicted by TargetscanHuman 7.1. Subsequently, the target genes were analysed by GO and KEGG pathway enrichment (Fig. 2a). GO term enrichment analysis showed the 379 genes enriched in “protein binding”, “negative regulation of transcription, DNA-templated”, and “transcription, DNA-templated”, etc. (Fig. 2b). KEGG pathway analysis showed the target genes of miR199a was significantly enriched in PI3K/Akt signalling pathway (Fig. 2c). Interestingly, SGK3, an important receptor in PI3K/Akt pathway, was suggested as potential targets of miR199a-mediated regulation (Fig. 2d). As a crucial molecular, SGK3 was enriched in several terms in GO and KEGG enrichment analysis (Fig. 2e), and the expression of SGK3 in liver cancer tissues was higher than that in adjacent non-tumour tissues (Fig. 2f). These results suggest that the PI3K/Akt signalling pathway was a target pathway of miR199a in HCCs.

miR199a could regulate Akt/mTOR pathway by targeting SGK3

To investigate the hypothesis that SGK3 is a key target molecule of miR199a in HCC, the potential binding sites of SGK3 3’UTR and miR199a was predicted. As shown in Fig. 3a, the miR199a potential binding sites on SGK3 3’UTRs was at position 2219–2225. Furthermore, qPCR and Western blot showed overexpression of miR199a significantly decreased SGK3 expression levels in HepG2 and HuH7 cells (Fig. 3b–d). A luciferase reporter assay was then carried out to determine if SGK3 was the direct target of miR199a in HEK293T cells. The 3′-UTR regions of SGK3 containing the predicted binding site of miR199a (SGK3-3ʹ-UTR-WT) or the mutant site (SGK3-3ʹ-UTR-Mut) were cloned into a luciferase vector. Luciferase activity results showed that overexpression of miR199a was able to decrease luciferase activity of SGK3-3ʹ-UTR-WT plasmid, but the effect disappeared in the SGK3-3ʹ-UTR-Mut, which indicated that the position 2219 to 2225 of SGK3 3’URT was the key binding site of miR199a (Fig. 3e). Further, the PI3K/Akt pathway was inhibited in miR199a mimics treated cells, such as the phosphorylation of AKT and mTOR were significantly repressed, as well as the proliferating cell nuclear antigen (PCNA) (Fig. 3f). These results suggested that miR199a represses the proliferation of HCCs by binding to SGK3 3′-UTR, regulating SGK3 expression, and inhibiting activation of the Akt/mTOR signalling pathway.

Down-regulation of SGK3 blocked the proliferation of HCCs through Akt/mTOR signalling pathway

To further investigate the functions of the miR199a target gene, SGK3, in HCC tumourigenesis, we constructed SGK3 stable knockdown cell lines of HepG2 and HuH7 using shRNA technology (Fig. 4a & 4b), as respected, the PI3k/Akt pathway was inhibited in shSGK3 cells, the phosphorylation of AKT and mTOR were repressed and the express level of PCNA was decreased by shRNA to SGK3 (Fig. 4b). CCK-8 assay indicated that the proliferation of stable knockdown cell lines was significantly suppressed compared with control cells (shRNA) (Fig. 4c & 4d). Moreover, the EdU uptake and EdU positive cells were also showed deficiency, indicated that the cell proliferation was inhibited (Fig. 4e & 4f). Thus, these results indicated that the effects of SGK3 on the proliferation of HCCs was mediated through the Akt/mTOR signalling pathway (Fig. 5).

miRNAs actively participate in basic cell processes and is abnormally expressed in many human cancers. Specifically, several miRNAs have been recently shown to be a crucial regulator in HCC development and progression, regulating processes including cell proliferation, viability, migration and invasion(Borel et al. 2012; Lou et al. 2019; Zhou et al. 2018). HCC is one of the leading causes of cancer mortality due to late diagnosis and limited treatment options. Discovery of novel molecular targets and pathways regulated by identified miRNAs in HCCs may improve our understanding of HCC biology and its treatment. HCC has been characterised with defective cell cycle and uncontrolled cellular proliferation(Yang et al. 2015). This study sought to elucidate the effects and the potential mechanism of miR199a in HCC. Herein, our results demonstrated that miR199a possibly acts as a tumour suppressor in HCC by targeting the SGK3-mediated Akt/mTOR pathway, which hopefully offers a potential therapeutic regimen for patients with HCC.

miR199a is located on chromosome 19 within intron 16 of the DNM2 gene. Emerging evidence is accumulating that miR199a is closely related to the occurrence and development of tumours(Qin et al. 2018; Gan et al. 2020). Liu X et al. proved that the expression level of miR199a in non-small cell lung cancer tissue was significantly lower than that in normal thyroid tissue, and was related to tumour invasion and metastasis(Liu et al. 2022). In addition, Ghosh et al. reported that the expression of miR-199a was also significantly inhibited in HCC(Ghosh et al. 2017). Thus, miR199a seems to be closely related to the occurrence and development of liver cancer. Moreover, it has been confirmed that hypoxia-suppressed miR-199a plays a decisive role in limiting glycolysis in HCC cells by targeting hexokinase-2 (Hk2) and pyruvate kinase-M2 (Pkm2)(Zhang et al. 2015). In the current study, our results showed that the level of miR199a was remarkably downregulated in HCC tissue and HCCs (HepG2 and HuH7). Furthermore, we found that upregulation of miR199a obviously inhibited the cell proliferation and reduced the PCNA expression in HCCs. These results together suggested that miR199a may function as a tumour suppressor during the development and progression of HCC.

A striking finding in this study was that miR199a suppressed the Akt/mTOR signalling pathway by targeting SGK3. SGK3 encoded by chromosome 8q12.2 is an important member of the serum and glucocorticoid-inducible protein kinase (SGK) family. SGK3 mediates a series of cellular processes, including cell proliferation and survival, and has been demonstrated to act as a downstream mediator of the PI3K signaling in tumourigenesis(Yao et al. 2018; Liu et al. 2018). Our present study showed that the target genes of miR199a were significantly enriched in the PI3K/Akt signalling pathway. SGK3, which is highly expressed in liver cancer tissues, may contribute to the aggressiveness and survival of liver cancer(Cao et al. 2019; Liu et al. 2020). Accumulating studies suggested that targeting SGK3 could lead to an inhibition of cell proliferation and migration of HCCs in vitro and in vivo. Our present study showed that miR199a directly targeted SGK3 in HCCs. Furthermore, miR199a overexpression or silencing of SGK3 obviously inhibited the PI3K/Akt pathway and the cell proliferation in HCCs, as confirmed by the reduced expression of PCNA and the decreased phosphorylation of both AKT and mTOR. The AKT/mTOR signalling pathway is known to regulate numerous physiological processes, including tumour cell-fate decisions and tissue homeostasis. Previous studies have established that the Akt/mTOR signalling plays an important role in the proliferation of HCCs and the development of HCC(Mello et al. 2020; Niture et al. 2020). Therefore, we suggested that miR199a inhibited the growth abilities of HCCs through targeting SGK3 by inactivating the Akt/mTOR signalling pathway (Fig. 5).

Taken together, miR199a was revealed to have the potential to inhibit the proliferation of HCCs in vitro through targeting SGK3. Furthermore, silencing of SGK3 inhibited the proliferation ability of HCCs by regulating Akt/mTOR signalling pathway. The finding of the present study indicated that miR199a exerted an antitumour effect on HCC development. Further study of the specific molecular mechanism of the miR199a/SGK3 axis will provide new hope for the cure or prevention of HCC.

Declaration of competing interest

The authors declare that there are no conflicts of interest.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the research grants from the National Natural Science Foundation of China (Grants number: 81901567), the Advantages Discipline Group (Biology and Medicine) Project in Higher Education of Hubei Province (No.2022BMXKQT2),the Natural Science Foundation of Hubei Province (2022CFB890), the Natural Science Foundation of Hubei Provincial Department of Education (Q20212105),Hubei Provincial Health Commission Research Project [grant numbers WJ2023F076],the Innovative Research Programme of Xiangyang No.1 People’s Hospital [grant numbers XYY2021D02],the Innovation and Entrepreneurship Training Program for College Students in the School of Pharmacy and Nursing, Hubei Medical University (Grants number: X202313249005), the “323” Public Health Project of the Hubei health commission and the Xiangyang No.1 People’s Hospital (grant number XYY2022-323).

Author Contribution

All authors contributed to the study conception and design. Material preparation, Experimental operation,Data collection and Analysis were performed by Ting Wu, Guilin Qi, Pan Liu,Jing Li and Min Guo.The the main manuscript text was written by HuiYu,Huidongfang Yang,Yuyin Jin,Wen Zhou and Wangheng Hu. Han Yu and Peng Duan was responsible for experimental design and paper revision. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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No competing interests reported.

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miR199a represses the proliferation of hepatocellular carcinoma cells by targeting SGK3 through Akt/mTOR pathway

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Abstract

Purpose

Methods

Results

Conclusion

Figures

Introduction

Materials and methods

Acquisition of microarray data

Patient samples

Cell culture and miRNA transfection

miRNA target prediction

Cell Counting Kit-8 (CCK-8) assay

5-Ethynyl-2'-deoxyuridine (EdU) incorporation assay

GO and KEGG analysis

Stable SGK3 knockdown cell line construction

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Western blot assay

Luciferase reporter assay

Statistical analyses

Results

miR199a was down-regulated in HCC tissue and cells lines

Overexpression of miR199a suppressed the proliferation of HCCs

PI3K/Akt signalling pathway was the miR199a target pathway

miR199a could regulate Akt/mTOR pathway by targeting SGK3

Down-regulation of SGK3 blocked the proliferation of HCCs through Akt/mTOR signalling pathway

Discussion

Declarations

Declaration of competing interest

Funding

Author Contribution

References

Additional Declarations

Supplementary Files

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