miR-183-5p attenuates the effect of sorafenib on human hepatocellular carcinoma via inhibiting SOCS6/JAK2/STAT3 pathway

doi:10.21203/rs.3.rs-3916655/v1

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miR-183-5p attenuates the effect of sorafenib on human hepatocellular carcinoma via inhibiting SOCS6/JAK2/STAT3 pathway

https://doi.org/10.21203/rs.3.rs-3916655/v1

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Objective:

MicroRNA plays a crucial role in the progression of hepatocellular carcinoma (HCC) and the resistance of HCC cells to sorafenib (SOR). Elevation of miR-183-5p is associated with poor survival among patients with HCC. This study aimed to investigate the impact of miR-183-5p on SOR resistance in HCC as well as its related signaling pathway. The objective is to provide new insights, directions, and a theoretical basis for the clinical diagnosis and treatment of HCC.

Design:

Human normal hepatocytes (LO2) and HCC cell lines (HepG2, Huh7, and MHCC97H) were cultured, and were constructed with miR-183-5p inhibition and SOCS6 overexpression. Biotrust analysis and qRT-PCR were employed to assess the expression of miR-183-5p in liver cancer tissues or cells, respectively. Flow cytometry determined apoptosis rate in each group of cells, while CCK was used for detecting the 50% inhibitory concentration (IC50) of HCC followed SOR treatment. Western blotting was used to detect protein expression changes of SOCS6, p-JAK2, JAK2, p-STAT3, and STAT3.

Results:

Bioinformatics revealed significantly high expression of miR-183-5p in liver cancer compared to normal tissues. Consistent with this analysis, the expression of miR-183-5p was upregulated in human HCC cell lines, in order of Huh7, HepG2, and MHCC97H, compared to that of non-HCC cells. CCK-8 assays results shown that the IC50 value of sorafenib in Huh7 cells with higher expression levels of miR-183-5p were more high than Hep3B and MHCC97H cells with the relative lower expression levels of miR-183-5p. SOCS6 was elevated with the miR-183-5p inhibition compared to the control. Furthermore, the IC50 value of sorafenib was significantly decreased following miR-183-5p inhibition and increased in the miR-183-5p overexpression compared to the mock treatment. Conversely, the IC50 value of sorafenib in the SOCS6 overexpression group was significantly decreased compared to the control.

Conclusions:

Dysregulation of the miR-183-5p-SOCS6/JAK2/STAT3 axis plays a critical role in patients' responses to SOR treatment. Manipulation of this axis could potentially enhance the survival of patients with HCC, especially in the context of addressing drug resistance.

Biological sciences/Biochemistry

Biological sciences/Cancer

Biological sciences/Cell biology

Biological sciences/Chemical biology

Biological sciences/Drug discovery

Human hepatocellular carcinoma

Sorafenib

miR-183-5p

Drug resistance

Hepatocellular carcinoma (HCC) stands as one of the most formidable malignancies globally^[1]. Surgical resection and liver transplantation emerge as frequent modalities for treating HCC, offering reasonable survival rates^[2]. Unfortunately, a quite significant number of HCC patients miss the window for these interventions due to the insidious onset and/or delayed diagnosis, resulting in an advanced disease stage upon initial identification. Consequently, conventional treatment regimens prove ineffective, relying solely on systemic medical therapy^[2]. Currently, drugs utilized for advanced HCC treatment encompass sorafenib (SOR), lenvatinib, and bevacizumab, with regorafenib and nivolumab as secondary options^[3]. Notably, SOR stands out as the most effective and first-line systemic drug for advanced HCC treatment^[2]. Hence, investigating the underlying mechanisms of SOR resistance and identifying novel therapeutic targets for HCC holds paramount significance in enhancing the prognosis of this condition.

SOR is a primary medication for advanced HCC, acting by inhibiting the activation of Ras/Raf/MEK/ERK and PI3K/AKT/mTOR pathways to impede tumor cell angiogenesis, proliferation, and apoptosis resistance^{[4, 5]}. Despite its efficacy, approximately 30% of HCC patients exhibit sensitivity, with high rates of resistance posing a significant impediment to its effectiveness^{[6, 7]}. Therefore, it is imperative to investigate the underlying mechanisms of sorafenib resistance, identify effective biomarkers to predict patient response to sorafenib treatment, and explore therapeutic agents that enhance SOR sensitivity, thereby optimizing its application.

MicroRNA (miRNA), a small non-coding RNA, exerts control over post-transcriptional gene expression by directly binding to the 3′ untranslated region of target mRNAs^[8], influencing approximately 60% of genes in the human genome^[8]. Emerging evidence underscores the pivotal regulatory role of miRNAs in the onset, progression, and development of drug resistance in malignant tumors^[9]. Particularly, involved that SOR regulated in HCC cells. miR-125b-5p was upregulated in HCC with SOR, and the 50% inhibitory concentration (IC50) of SOR in HCC was significantly increased after overexpression of miR-25b-5p, indicating that miR-125b-5p can promote the HCC to SOR^[10]. Not only that, miR-183-5p has been identified as a potential oncogene associated with various processes, including proliferation, apoptosis, and metastasis, especially resistance^{[11, 12]}. Recent studies indicate that elevated miR-183-5p expression levels correlate with poor survival in HCC patients^[12]. Our studies have verified that miR-183-5p promoted HCC migration and invasion via increasing aerobic glycolysis through targeting PTEN/Akt/mTOR signaling^[13]. Furthermore, it has been confirmed that miR-183-5p contributes to 5-FU resistance in HCC^[14]. However, the potential involvement of miR-183-5p in sorafenib resistance remains to be established. Unraveling the mechanisms by which miR-183-5p contributes to the development of drug resistance may pave the way for the exploration of novel approaches in HCC therapy.

Suppressor of cytokine signaling 6 (SOCS6) belongs to the negative regulators of the cytokine family and growth factor signaling SOCS, featuring two highly conserved functional domains: SH2 domains and C-terminal SOCS boxes. The SOCS box domain includes two central interaction sites, BC-box and Cul-box^[15]. Furthermore BC-box in SOCS6 enhances the ubiquitination of Janus kinase 2 (JAK2), inhibited the JAK2/STAT3 signaling pathway, promoted the differentiation in epidermal stem cells^[16]. JAK2, a non-receptor tyrosine kinase, is activated by phosphorylation of STAT3 protein^[17]. Signal transducer and activator of transcription 3 (STAT3) serves as a pivotal oncogenic transcription factor, often associated with poor prognosis and chemotherapy resistance in various cancers^{[18, 19]}. Crucially, activationof STAT3 is linked to sorafenib-induced cell death resistance ^[20]. Notably, studies reveal an upregulation of STAT3 in HCC^[21]. While, Downregulation of SOCS6 expression in HCC^[22] is correlating with chemoresistance^[23]. This is implied that SOCS6 emerges as a key regulator of sorafenib-induced HCC cell death.

SOCS6 is a potential target gene of miR-183-5p, which has been validated in hepatocellular carcinoma and pancreatic cancer^[24–26]. SOCS6 overexpression and miR-183 knockdown significantly increased the sensitivity of HCC cells to 5-FU, which thought MiR-183 of overexpression abrogated the effect of SOCS6 in enhancing 5-FU sensitivity^[14]. Other research found overexpression of SOCS6 could significantly lower HCC cells growth and invasion, while increase apoptotic^[27]. In fact, miR-21 and miR-183 can modulate HCC cells via simultaneously target SOCS6^[28]. However, It is blank on whether miR-183-5p regulates sorafenib resistance in HCC, thus the intricate correlation and feedback loop between the two necessitate further exploration. In the current study, our results indicate that miR-183-5p enhances HCC cells to resistance of sorafenib by targeting the SOCS6-mediated JAK2/STAT3 signaling pathway.

Cell Culture

Three HCC cell lines and a human normal hepatic cell line (LO2) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HCC cell lines (HepG2, Hep3B, and Huh7) were obtained from The Second People's Hospital of Lanzhou University (Gansu, China)^[29]. These HCC cell lines were cultured in DMEM (HyClone Laboratories, Logan, UT, USA), while the LO2 cell line was maintained in RPMI 1640 medium. All culture media were supplemented with 10% fetal bovine serum (Yeasen, Shanghai, China) and 1% ampicillin–streptomycin (Solarbio, Beijing, China). The cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂.

Cell Transfection

miRNA mimics, and their respective double-stranded negative controls (NC), as well as, miRNA inhibitors and their single-stranded scrambled controls (NC inhibitor), were obtained from GenePharma RiboBio (Ribobio Co., Guangzhou, China). The sequences for all were listed in Table 1. For the modulation of miR-183-5p expression in HCC cells, miR-183-5p mimic and miR-183-5P. Inhibitor were employed, confirmed through qPCR. Transfection of all siRNAs, miRNA mimics, miRNA inhibitors, and their controls into cells was conducted, using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions.

Table 1

Gene Sequences
Reagent	Sequence
hsa-miR-183-5p inhibitor inhibitor NC hsa-miR-183-5p mimics mimics NC	5'-AGUGAAUUCUACCAGUGCCAUA-3' 5'-CAGUACUUUUGUGUAGUACAA-3' sense: 5'-UAUGGCACUGGUAGAAUUCACU-3' antisense: 5'-UGAAUUCUACCAGUGCCAUAUU-3' sense: 5'-UUCUCCGAACGUGUCACGUTT-3' antisense: 5'-ACGUGACACGUUCGGAGAATT-3'

Table. 1: The gene sequences of miRNA mimics, and their respective double-stranded negative controls (NC), miRNA inhibitors and their single-stranded scrambled controls (NC inhibitor).

Cell viability assay

Cell viability was assessed using the CCK-8Kit-8 (Dojindo, Kumamoto, Japan) following the manufacturer's instructions. HCC cells (8 x 10³) were seeded into 96-well plates with conditioned media containing various concentrations of sorafenib for the specified durations. After a 2-hour incubation with CCK-8 solution, the absorbance of the wells was measured using an MRX II microplate reader (450 nm, Dynex, Chantilly, VA, USA). Relative cell viability was calculated as a percentage of untreated control HCC cells.

Quantitative Real-Time Polymerase Chain Reaction (qPCR)

Total RNA was extracted from in vitro cultured liver cancer cells using the Trizol kit. The reverse transcription kit was employed for cDNA synthesis, and subsequently, primers targeting βactin and U6 were utilized as internal references for amplification. The reaction system comprised 20 µl, and the reaction conditions were set at 94°C for 20 s, 60°C for 35 s, with a total of 40 reaction cycles. The relative mRNA expression was determined using the 2-△△Ct method.

Western Blot

The concentration of proteins, extracted from cultured cells using RIPA lysate, was determined with a BCA kit. Then the proteins were run on a 10% SDS-PAGE gel and transferred to a PVDF membrane. The PVDF membrane underwent blocking in TBST buffer for two hours, as described Begum H^[30]. Subsequently, the membrane was incubated at 4°C overnight with specific primary antibodies (SOCS6, STAT3, and the internal reference GAPDH). Finally, the ECL reagent was applied for exposure, and the Gel-Pro-Analyzer software was utilized for measuring the gray value. The relative expression level of the protein was determined by the ratio of the gray value of the target protein to the gray value of the internal reference protein.

Flow Cytometry

The liver cancer cells were collected after an overnight culture in DMEM medium with the respective treatments. Subsequently, the harvested cells were resuspended in 100 µm annexin V binding buffer (Dojindo, Kumamoto, Japan) supplemented with 1% annexin V-FITC. Flow cytometry analysis was then performed on these cells using BD FACSCanto II (NJ, USA)^[31].

Statistical Analysis

All data were subjected to statistical analysis using SPSS 25.0 software, with a significance level set at 0.05. Quantitative data descriptions were assessed for normality, and those following a normal distribution were presented as mean ± standard deviation (Means ± S.E.M), while those not adhering to normal distribution were represented by the median and interquartile range. For comparisons between the means of two groups adhering to normal distribution with homogenous variance, the independent sample t-test was employed. In cases involving multiple groups, the one-way ANOVA test was utilized. Bivariate subjects conforming to normal distribution underwent Pearson correlation analysis. Conditional logistic regression analysis was applied to evaluate the expression levels of miR-183-5p and related factors in each hepatocellular carcinoma cell line. Graphs depicting the data were generated using Graphpad Prism 5.0 software.

1. Upregulated MiR-183-5p in HCC was associated with SOR resistance.

We first downloaded the miRNA expression data of liver cancer tissue specimens (370) and adjacent cancer tissue samples (50) from the TCGA database (http: //www. targetscan.org/)., and analyzed the difference between the expression of miR-183-5p (Fig 1A, B), which showed that the expression of miR-183-5p in liver cancer tissues was significantly higher than that of normal tissues. Then we analyzed the expression of miR-183-5p in three HCC cell lines and normal hepatic cells by RT-qPCR. The results revealed significantly upregulated expression of miR-183-5p in HCC cell lines compared with the normal hepatic cells (Fig. 1C). In addition, we showed differences in the expression levels of miR-183-5p between three HCC cell lines (MHCC97H, HepG2 and Huh-7) and found that Huh-7 cells expressed higher levels of miR-183-5p than Hep3B and MHCC97H cells. We further analyzed the relationship between miR-183-5p and sorafenib resistance of HCC. CCK-8 results shown that Huh7 cells with higher expression levels of miR-183-5p were more resistant to sorafenib than Hep3B and MHCC97H cells with the lower expression levels of miR-183-5p (Fig. 1D). Based on these data, miR-183-5p expression is significantly correlated with sorafenib resistance of HCC.

Initially, we retrieved miRNA expression data for 370 liver cancer tissue specimens and 50 adjacent cancer tissue samples from the TCGA database (available at http://gepia.cancer-pku.cn/ ). A substantial upregulation of 183-5p expression was obtained in liver cancer tissues compared to non-cancer tissue (Fig 1A, B). Subsequently, it was assessed miR-183-5p expression in three HCC and normal hepatic cell lines, using RT-qPCR, demonstrating 1.9-fold upregulation of miR-183-5p in HCC cell lines compared to normal hepatic cells (p<0.05) (Fig. 1C). We also observed that upregulation of miR‑183‑5p was in order of Huh-7, Hep3B and MHCC97H cells. Additionally, it was analyzed that the correlation between miR-183-5p and sorafenib resistance in HCC, showing that there was correlation between miR-183-5p and sorafenib resistance in HCC, i.e. the higher miR-183-5p, the more resistance of SOR from these HCC cell lines (Fig. 1D).

Fig. 1: miR-183-5p Upregulation in Human HCC Cell lines correlates with SOR resistance. Bioinformatics support miR183 was higher in HCC than the control, which was consistent with our finding that upregulated in three HCC cell lines. Additionally, a correlation between miR183 expression and SOR resistance. Our data suggest miR183 plays an important role in the pathogenesis of HCC, particularly relating to the SOR sensitivity during the treatment.

A. Overall difference analysis of miR-183-5p expression between HCC (n=370) and adjacent non-HCC tissue (n=50), ***P<0.001.

B. Pairwise comparison of miR-183-5p expression between HCC (n=50) and adjacent non-HCC tissue (n=50), ***P<0.001.

C. Relative expression of miR-183-5p mRNA, assessed by the RT-qPCR method in non-HCC and HCC cell lines (n=3), **P<0.01, ****P<0.0001.

D. SOR IC50 detection using the CCK-8 assay in HCC cell lines (MHCC97H, HepG2, and Huh7).

2. miR-183-5p promotes SOR resistance in hepatoma cells

To determine the potential role of miR-183-5p in SOR resistance, it was manipulated the expression of miR-183-5p in HCC cell lines using transfection (Fig. 2A). The miR-183-5p mimic transfected HCC cells increased SOR resistance, supported by a decrease in the 50% inhibitory concentration (IC50). Conversely, miR-183-5p inhibitor transfected HCC cells increased sensitivity to SOR, reflected in an elevated IC50 (Fig. 2B). miR-183-5p mimic significantly lower apoptosis rates in HCC cells following SOR treatment, compared to that in the miR-NC and untransfected groups, using flow cytometric analysis (Fig. 2C).

Fig. 2: miR-183-5p promotes SOR Resistance in HCC Cells.MiR183 inhibitor enhanced SOR sensitive, but mimics reduced the sensitive from the different HCC cell lines. This is in line with our findings above, suggesting the miR183 is a good therapeutic target molecule in responses to SOR treatment.This is further supported by the apoptotic rate from HCC cells, i.e. the higher miR183 and the lower apoptosis, consistent with drug resistance.

A. qRT-PCR analysis of the transfection efficiency of miR-183-5p mimics and inhibitor in HCC, ****P<0.0001.

B. CCK-8 detection of the sorafenib IC50 after transfection with miR-183-5p mimics and inhibitor.

C. Flow cytometry analysis of sorafenib-induced apoptosis rates after transfection with miR-183-5p mimics and inhibitor, ****P<0.0001.

3. miR-183-5p targets SOCS6 in HCC cell lines

Specific gene targets of miR-183-5p were identified using TargetScan, a publicly accessible miRNA prediction database (http://www.targetscan.org/). From the multitude of predicted targets, SOCS6, recognized as a tumor suppressor associated with drug resistance, was singled out (Fig. 3A). Interestingly, the levels of SOCS6 mRNAs and proteins increased with the miR-183-5p inhibitor but remained unchanged in HCC cells following miR-183-5p overexpression (Fig. 3B, C). These findings affirm that SOCS6 is a direct target of miR-183-5p, suggesting a potential link between miR-183-5p and sorafenib resistance in HCC cells.

Fig. 3 miR-183-5p targets SOCS6 in HCC cell lines. A.

Bioinformatics suggesting that SOCS6 is a target for miR183 in HCC. We validated that miR inhibitor enhanced the production of SOCS6, but miR mimics showed no significant change of SOCS6. Our data suggest that SOCS6 is a target for miR following the inhibitor. However miR183 wasn’t significantly modified following the mimics, we speculate it may be attributed to the fact that miR183 was saturated in the HCC cells, and no add on effect.Target gene prediction map. B. SOCS6 mRNA expression levels were detected by RT-qPCR after transfection of miR-183-5p mimics and inhibitor, *P<0.05, **P<0.01. C. SOCS6 protein expression levels were detected by western blot after transfection of miR-183-5p mimics and inhibitor, ****P<0.0001.

4. SOCS6 is a key factor in SOR-induced death of hepatoma cells

To investigate the impact of SOCS6 on SOR-induced death in HCC cells, a HCC cell line overexpressing SOCS6 was generated (Fig. 4A). Our findings demonstrated that HCC cells with overexpressed SOCS6 were more sensitive to SOR than the control, as evidenced by an increase in the IC50 (Fig. 4B). The apoptosis rate in the HCC cells overexpressing SOCS6 with SOR treatment significantly surpassed the apoptosis rate induced in the control (Fig. 4C). These results affirm the pivotal role of SOCS6 in sorafenib-induced death of liver cancer cells, highlighting that overexpression of SOCS6 promotes drug resistance in HCC against SOR.

Fig. 4: SOCS6 in SOR-Induced Death of HCC.

A. Transfection efficiency of SOCS6 in HCC was assessed through RT-qPCR and western blot analysis, ****P<0.0001.

B. CCK-8 assay illustrating sorafenib IC50 after the overexpression of SOCS6.

C. Flow cytometry analysis of the sorafenib-induced apoptosis rate following SOCS6 overexpression, ***P<0.001, ****P<0.0001.

5. miR-183-5p inhibits the JAK2/STAT3 Pathway by targeting SOCS6

To elucidate potential interactions among miR-183-5p, SOCS6, and JAK2/STAT3, it was determined that the expression of SOCS6, p-JAK2, JAK2, p-STAT3, and STAT3 proteins, using Western blot following miR-183-5p inhibition and overexpression. A significant decrease (how much?) in the relative expression of p-JAK2 and p-STAT3 proteins following miR-183-5p inhibition, while the relative expression of JAK2 and STAT3 proteins remained unchanged. Conversely, there were no significant changes in p-JAK2, p-STAT3 proteins, JAK2, and STAT3 proteins after miR‑183‑5p overexpression (Fig. 5A, B). Furthermore, SOCS6 exhibited a similar effect after either overexpression and inhibition of miR-183-5p (Fig. 5C). These findings suggest that miR-183-5p may enhance the JAK2/STAT3 signaling pathway by inhibiting SOCS6.

Fig. 5: miR-183-5p Inhibits the JAK2/STAT3 Pathway by Targeting SOCS6.

A, B. Western blot analysis of SOCS6, phospho-JAK2, JAK2, phospho-STAT3, and STAT3 expression in HCC cells after transfection with miR-183-5p mimics and inhibitor, ****P<0.0001.

C. Western blot analysis of SOCS6, phospho-JAK2, JAK2, phospho-STAT3, and STAT3 expression in HCC cells following overexpression of SOCS6, ***P<0.001, ****P<0.0001.

As a highly lethal malignant tumor, hepatocellular carcinoma (HCC) poses a significant threat to human health. SOR, a polykinase inhibitor, is a well-established targeted drug for treating advanced HCC. By targeting c-Kit, VEGFR, PDGFR-β, and other tyrosine kinases, SOR inhibits the activation of Ras/Raf/MEK/ERK and PI3K/AKT/mTOR signaling pathways, thus impeding tumor cell angiogenesis, proliferation, and apoptosis resistance ^{[5, 6]}. Despite its efficacy in ~ 30% of patients, high rates of resistance remain a substantial obstacle^[7]. Therefore, investigating the mechanisms of sorafenib resistance, identifying effective biomarkers for predicting patient response, and exploring agents to enhance sorafenib sensitivity are critical endeavors.

MicroRNAs (miRNAs) have emerged as promising biomarkers and therapeutic targets for sorafenib sensitivity and clinical prognosis, potentially reversing HCC cell resistance to sorafenib ^{[32, 33]}. Notably, miR-183-5p has been implicated in drug resistance in various cancers, showing that altering miR-183-5p expression levels can influence drug sensitivity in laryngeal and prostate cancers^{[34, 35]}. In our investigation, we observed significantly elevated miR-183-5p expression in HCC cells, promoting the proliferation and apoptosis resistance of HCC cells, predicting an unfavorable response to SOR treatment. Conversely, low miR-183-5p expression correlated with increased SOR sensitivity. Therefore, we propose that miR-183-5p levels could serve as a reliable indicator for predicting SOR sensitivity in HCC patients, with miR-183-5p inhibition potentially acting as a sensitizer for SOR treatment.

Further elucidating the mechanism of miR-183-5p involvement in HCC SOR resistance, we identified SOCS6 as a downstream target through bioinformatics analysis, consistent with findings in HCC and pancreatic cancer ^{[14, 36, 37]}. SOCS6 is often downregulated in various cancers ^[22]. Our study revealed that overexpressing SOCS6 enhanced SOR sensitivity in HepG2 and Huh7 cells, leading to increased apoptosis. Importantly, we established a link between miR-183-5p and SOCS6, demonstrating that low miR-183-5p expression promotes SOCS6 transcription and translation. Surprisingly, high miR-183-5p expression did not affect SOCS6 levels significantly, potentially due to saturation of miR-183-5p binding sites on SOCS6 in highly expressing HCC cells.

The JAK2/STAT3 signaling pathway, a common mediator of cytokine signals, is negatively regulated by the SOCS family. Given SOCS6's role as a JAK2/STAT3 pathway inhibitor ^{[17, 38]}, we explored the possibility of miR-183-5p influencing this pathway in hepatoma cells. Our findings suggest that low miR-183-5p expression increased SOCS6 protein levels, decreased p-JAK2 and p-STAT3 levels, and left total JAK2 and STAT3 protein levels unchanged. Overexpressing SOCS6 replicated these effects, supporting its role as a JAK2/STAT3 pathway inhibitor. Unexpectedly, miR-183-5p overexpression had no significant impact on protein levels, suggesting that miR-183-5p's effect on HCC sorafenib resistance may involve alternative pathways.

In conclusion, our study suggests that higher lever of miR-183-5p may promote HCC resistance to SOR by inhibiting SOCS6 futher activating the JAK2/STAT3 signaling pathway. Other pathways which miR-183-5p influences SOR resistance in HCC need to uncover.

Compliance with Ethical Standards

The study has received approval from the Ethics Committee of Gansu University of Chinese Medicine and adheres to laboratory safety standards. It will not be submitted for publication elsewhere. All authors declare no conflicts of interest related to the publication of this paper.

Author Contribution

All authors have no conflict of interest regarding the publication of this paper.

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miR-183-5p attenuates the effect of sorafenib on human hepatocellular carcinoma via inhibiting SOCS6/JAK2/STAT3 pathway

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Abstract

Objective:

Design:

Results:

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INTRODUCTION

METHODS

Cell Culture

Cell Transfection

Cell viability assay

Quantitative Real-Time Polymerase Chain Reaction (qPCR)

Western Blot

Flow Cytometry

Statistical Analysis

RESULTS

DISCUSSION

Declarations

Compliance with Ethical Standards

Author Contribution

References

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Supplementary Files

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