MicroRNA‐216b suppresses the cell growth of hepatocellular carcinoma by inhibiting Ubiquitin‐specific peptidase 28 expression

Abstract

MicroRNA‐216b (miR‐216b) has been reported to be downregulated in several tumors, its mechanism is still little‐studied in hepatocellular carcinoma (HCC). In the present study, we found that miR‐216b was downregulated in HCC, but Ubiquitin‐specific peptidase 28 (USP28) was upregulated. In addition, Kaplan‐Meier‐plotter analysis indicated that liver cancer patients with high miR‐216b expression had a longer overall survival, but patients with high USP28 had a shorter overall survival. Further studies showed that overexpression of miR‐216b inhibited HCC cell growth, and molecular investigations revealed that miR‐216b targeted USP28 and inhibited its expression in HCC cells. In addition, overexpression of miR‐216b suppressed the substrates' expression of USP28, for example, c‐Myc, and miR‐216b overexpression also inhibited Cyclin E expression as well as upregulating p27 expression, both of which were the downstream signals of c‐Myc. These results indicated that miR‐216b downregulated USP28/c‐Myc signaling in HCC cells. Collectively, this study demonstrated that miR‐216b/c‐Myc axis could be as a potential target for HCC therapy in the future.

1. INTRODUCTION

Liver cancer is one of the most common malignancies in China, and our country is one of the countries with high incidence of liver cancer in the world.1, 2 Liver cancer can be divided into two main categories in clinic: metastatic liver cancer and primary liver cancer.3 In clinic, hepatocellular carcinoma (HCC) is more common in primary liver cancer, and the clinical treatments of HCC mainly include surgery, target‐drug therapy, radiofrequency ablation, interventional therapy, and immunotherapy.4, 5 Among these, surgery is still the first choice if conditions permitted.6 Although the levels of diagnosis and treatments of HCC have improved significantly in recent years, most patients with late stages are still untreatable.7 Thus, identifying new pathogenesis is very important for the treatment of HCC.

Ubiquitin‐specific peptidase 28 (USP28) is a deubiquitinase, which has been found upregulated in several tumors, and displays its function as an oncoprotein.8 In breast cancer and colon cancer, USP28 was found highly expressed.9 As a deubiquitinase, USP28 bound to c‐Myc protein through an interaction with E3 ligase FBW7α, and USP28 stabilized c‐Myc by decreasing its polyubiquitination, which was essential for cell proliferation in tumors.9 It has also been reported that USP28 knockdown enhanced the radiosensitivity of esophageal cancer cells by destabilizing c‐Myc protein and enhancing the accumulation of HIF‐1α.10 In non‐small cell lung cancer (NSCLC), USP28 was upregulated and predicted a poor index of NSCLC patients.11 In mechanism, USP28 bound to STAT3, and stabilized STAT3 by decreasing its polyubiquitination.11 Thus, inhibiting USP28 could be as a promising strategy for tumor diagnosis or treatment.

In the present study, we found that microRNA‐216b (miR‐216b) was markedly decreased in HCC cell lines, and was negatively correlated with USP28 expression. Further studies showed that miR‐216b targeted USP28 and inhibited its expression in HCC cells. Moreover, overexpression of miR‐216b suppressed HCC cell growth, but USP28 overexpression reduced miR‐216b‐induced cell growth inhibition. Targeting miR‐216b/USP28 axis could be as a potential strategy for the diagnosis and treatment of HCC in clinic.

2. MATERIALS AND METHODS

2.1. Cells and cell culture

Four HCC cell lines (BEL‐7404, HepG2, Huh‐7, and SNU‐387) and one normal liver cell line L‐02 were purchased from ATCC (Manassas, Virginia). All HCC cell lines and the normal liver cell line were cultured in RPMI 1640 medium (Hyclone) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. HCC patient samples were collected from Songshan Hospital of Qingdao University. The collection and use of human tissues were approved by the Institutional Review Board of Songshan Hospital of Qingdao University.

2.2. Quantitative real‐time polymerase chain reaction

The quantitative real‐time polymerase chain reaction (qRT‐PCR) was carried out to analyze miR‐216b expression in HCC cells. MiRNA qRT‐PCR primer sets specific for miR‐216b were synthesized by GenePharm (Suzhou, China). The MiRNeasy Mini Kit (QIAGEN, Germany) was used to extract total RNA, and the miRNA bulge‐loop was reverse‐transcribed with the Quantscript RT Kit (QIAGEN, Germany). MiRNAs were normalized against U6 snRNA.

To analyze the mRNA levels of USP28, qRT‐PCR was carried out by using SYBR Green qPCR Master Mix (Bimake, Houston, Texas). β‐actin was used as an internal control. Primers used in this part were as follows: USP28, forward 5′‐ TCTGGATGCCATTGAGGT ‐3′ and reverse 5′‐ TAGGGAGAATTGGGTCGA ‐3′; β‐actin, forward 5′‐ GGAAATCGTGCGTGACATT ‐3′ and reverse 5′‐ CAGGCAGCTCGTAGCTCTT ‐3′.

2.3. Cell growth and viability

HCC cell lines were transfected with microRNAs or plasmids for indicated time, and the cell viability was analyzed by Cell Counting Kit‐8 (CCK‐8) assay as stated by the manufacturer's instructions (Bimake, Houston, Texas).

2.4. Luciferase assay

The binding sites of miR‐216b in USP28 3'UTR were predicted online (http://www.microrna.org), and fragments with predicted miR‐216b binding sites or the mutants were amplified by PCR and subcloned into pGL3 plasmids. Then, above‐mentioned recombinant plasmids together with miR‐216b or miR‐NC were transfected into HepG2 cells for 72 hours, followed by luciferase assay (Promega, Madison, Wisconsin) as stated by the manufacturer's instruction.

2.5. Immunoblotting

To analyze the protein expression, immunoblotting analysis was carried out as described previously.12 The primary antibodies against USP28, β‐actin, c‐Myc, Cyclin E, and p27 were purchased from CST (Cell Signaling Technology, Danvers, Massachusetts). Anti‐rabbit and anti‐mouse IgG horseradish peroxidase conjugated antibodies were purchased from CST.

2.6. MiRNA, plasmids construction, and transfection

The negative control (miR‐NC) and miR‐216b mimic (Gene ID: MIMAT0004959) were synthesized by GenePharm (Suzhou, China). The human USP28 gene was amplified by PCR, and subcloned into pcDNA3.1 plasmids. HCC cells were transfected with 50 nmol/L miR‐216b mimic or miR‐NC by LipofectamineRNAiMAX Reagent (Invitrogen), and relative plasmids were transfected into HCC cells by using Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions.

2.7. Kaplan‐Meier plotter analysis

To analyze the overall survival of HCC patients with low or high miR‐216b, Kaplan‐Meier (KM) Plotter was used online (http://kmplot.com) based on the liver cancer miRNA database by selecting the non‐commercial spotted platform,13 and patients were split by auto select best cutoff, and cutoff value used in analysis was 1.19. To analyze the overall survival of HCC patients with low or high USP28 expression, KM Plotter was used by Pan‐cancer RNA‐seq database, and patients were split by auto select best cutoff, and cutoff value used in analysis was 265.

2.8. Statistical analysis

To compare two groups in the study, student's t test was used. To compare multi‐groups, ANOVA test was used. The statistical significance was defined as a P value less than .05.

3. RESULTS

3.1. MiR‐216b is downregulated and negatively associated with USP28 expression in HCC

Firstly, four HCC cell lines and one normal liver cell line were collected for qRT‐PCR analysis, and the results showed that miR‐216b was significantly downregulated in HCC cell lines compared with the normal liver cell line (Figure 1), but USP28 was markedly upregulated (Figure 1). In addition, the correlation analysis showed that miR‐216b expression was negatively correlated with USP28 expression in HCC cell lines (Figure 1). Moreover, miR‐216b was decreased but USP28 was elevated in HCC tumor tissues (Figure 1), and miR‐216b expression was also negatively correlated with USP28 in HCC tumor tissues (Figure 1). In addition, the public cancer database KM Plotter showed that liver cancer patients with high miR‐216b had a longer overall survival (Figure 2), but patients with high USP28 had a shorter overall survival (Figure 2). These results indicated that miR‐216b was negatively associated with USP28 in HCC.

Figure 1.

MicroRNA‐216b (miR‐216b) is negatively associated with USP28 in HCC. A. Normal liver cell line showed higher relative expressions of miR‐216b than four cancer cell lines. qRT‐PCR was used to detect miR‐216b expression. B, Four HCC cell lines showed higher relative expressions of USP28 than the normal. qRT‐PCR was used to detect USP28 expression. C, Correlation analysis for Figure 1. D and E, MiR‐216b was decreased but USP28 was elevated in HCC tumor tissues. Twelve pairs of paracancerous and tumor tissues of HCC were prepared for qRT‐PCR against miR‐216b (D) and USP28 (E). F, Correlation analysis for Figure 1. **P < .01. HCC, hepatocellular carcinoma; qRT‐PCR, quantitative real‐time polymerase chain reaction; USP28, Ubiquitin‐specific peptidase 28

Figure 2.

MicroRNA‐216b (miR‐216b) predicts a positive index but USP28 predicts a negative index for patients with HCC. A and B, The overall survival of HCC patients with low or high expression of miR‐216b (A) or USP28 (B) was analyzed by KM plotter (http://kmplot.com). HCC, hepatocellular carcinoma; USP28, Ubiquitin‐specific peptidase 28

3.2. Overexpression of miR‐216b inhibits HCC cell growth

Subsequently, to confirm the function of miR‐216b in HCC, miR‐216b mimics were used to overexpress miR‐216b in HepG2 and SNU‐387 cells (Figure 3). As shown in Figure 3, miR‐NC or miR‐216b mimic was transfected into HepG2 or SNU‐387 cells for indicated times, followed by CCK‐8 assay. In addition, the CCK‐8 assay showed that overexpression of miR‐216b could significantly suppress the cell growth of HepG2 (Figure 3) and SNU‐387 (Figure 3) cells. For example, at Day 5, the cell survival rate of HepG2 cells transfected with miR‐216b mimic had nearly a 25% decrease than the control (Figure 3), and in SNU‐387 cells, there was also about a 25% decrease in the cell survival rate of cells transfected with miR‐216b mimic (Figure 3). Thus, these results revealed that miR‐216b overexpression inhibited HCC cell growth.

Figure 3.

Overexpression of MicroRNA‐216b (miR‐216b) inhibits HCC cell growth. A, MiR‐216b was overexpressed in HepG2 and SNU‐387 cells. Cells were transfected with miR‐NC or miR‐216b mimic for 72 hours, followed by qRT‐PCR. B and C, MiR‐216b inhibited HCC cell growth. MiR‐NC or miR‐216b mimic was transfected into HepG2 (B) and SNU‐387 (C) cells for indicated times, followed by CCK‐8 assay at days 0, 1, 3, and 5. *P < .05; **P < .01

3.3. MiR‐216b targets USP28 and inhibits its expression in HCC cells

To further investigate whether miR‐216b targeted USP28, a public miRNA targets prediction website was used. As shown in Figure 4, miR‐216b was predicted to bind to the 3'UTR of USP28. Then, to confirm this result, a mutant of USP28 3'UTR was generated (Figure 4, lower), and miR‐216b or miR‐NC along with wild‐type (WT) or mutated USP28 3'UTR were transfected into HepG2 cells for 72 hours, followed by luciferase assay (Figure 4). As shown in Figure 4, miR‐216b markedly suppressed WT USP28 3'UTR‐driven luciferase activity, but had no significant effect on mutated USP28 3'UTR‐driven luciferase activity, which further suggested miR‐216b targeted USP28. Subsequently, HCC cells were also transfected with miR‐216b mimics to detect the mRNA and protein level of USP28, and the results showed that overexpression of miR‐216b could obviously inhibit the mRNA and protein level of USP28 in HCC cells (Figure 4). Above‐mentioned results suggested that miR‐216b targeted USP28 and inhibited its expression in HCC cells.

Figure 4.

MicroRNA‐216b (miR‐216b) targets USP28 and inhibits its expression in HCC cells. A, The binding sites of miR‐216b in USP28 3'UTR were predicted online (http://www.microrna.org). B, MiR‐216b suppressed USP28 3'UTR‐driven luciferase activity. MiR‐216b or miR‐NC along with wild‐type (WT) or mutant (Mut.) USP28 3'UTR was transfected into HepG2 cells for 72 hours, followed by luciferase assay. C and D, MiR‐216b inhibited USP28 expression. HepG2 and SNU‐387 cells were transfected with miR‐NC or miR‐216b mimic for 72 hours, followed by qRT‐PCR (C) or immunoblotting (D). **P < .01; n.s. means non‐sense

3.4. MiR‐216b downregulates USP28/c‐Myc axis in HCC cells

It has been reported that c‐Myc was a substrate of USP28, and USP28 could enhance c‐Myc signaling by decreasing its ubiquitin‐proteasome degradation.9 What we found earlier, miR‐216b targeted USP28 and inhibited its expression in HCC cells. Then, in this section, we wanted to know whether miR‐216b regulated USP28/c‐Myc signaling in HCC cells. As shown in Figure 5, overexpression of miR‐216b could obviously inhibit both of USP28 and c‐Myc expressions in HCC cells. In addition, our further studies also showed that miR‐216b overexpression downregulated Cyclin E expression, but upregulated p27 expression, both of which were the downstream signals of c‐Myc (Figure 5). In addition, overexpression of c‐Myc could also significantly rescue miR‐216b‐induced cell growth inhibition in both of HepG2 (Figure 5) and SNU‐387 cells (Figure 5). Thus, above‐mentioned studies revealed that miR‐216b downregulated USP28/c‐Myc signaling in HCC cells.

Figure 5.

MicroRNA‐216b (miR‐216b) downregulates USP28/c‐Myc axis in HCC cells. A, MiR‐216b inhibited c‐Myc and Cyclin E expression, but upregulated p27. HepG2 and SNU‐387 cells were transfected with miR‐NC or miR‐216b mimic for 72 hours, followed by immunoblotting. B and C, C‐Myc overexpression rescued miR‐216b‐induced cell growth inhibition. Myc‐USP28 plasmids or miR‐216b mimics were transfected into HepG2 (B) or SNU‐387 (C) cells for 72 hours, followed by CCK‐8 assay. In addition, the transfection efficiency of Myc‐USP28 plasmids was identified by immunoblotting. **P < .01

4. DISCUSSION

In the present study, we found that miR‐216b was markedly downregulated in both of HCC cell lines and tumor tissues, and miR‐216b was also predicted as a positive index for liver cancer patients by KM Plotter. It is known that aberrant expression of microRNAs is closely associated with the progression of most tumors.14 In addition, our present study was also consistent with previous studies. One previous study showed that miR‐216b was downregulated in HCC, and ectopic expression of miR‐216b suppressed HCC cell growth by targeting FOXM1.15 In breast cancer, miR‐216b was found decreased and overexpression of miR‐216b inhibited cell proliferation and progression.16 In pancreatic cancer, miR‐216b was also found downregulated, and miR‐216b overexpression inhibited cell progression and promoted apoptosis by downregulating KRAS.17 These studies indicated that miR‐216b served as a tumor suppressor in tumors, including HCC.

Although altered expression of miR‐216b has been reported in HCC, but its functional mechanisms have not been fully investigated. At present study, we firstly found that miR‐216b expression was negatively correlated with USP28, which was a deubiquitinase. In addition, overexpression of miR‐216b suppressed HCC cell growth, but this effect could be attenuated by ectopic expression of USP28. Our further studies demonstrated that miR‐216b targeted USP28 3'UTR and inhibited the expression of USP28 in HCC cells. In addition, miR‐216b overexpression downregulated c‐Myc protein and its downstream signals by inhibiting USP28 expression in HCC cells. It is known that miRNAs have several targets in different tumors.18 Thus, USP28 could be as a novel target of miR‐216b in HCC. Interestingly, a paper reported that miR‐92b‐3p bound to USP28 3'UTR and inhibited its expression in pulmonary artery smooth muscle cells,19 which further suggested that different miRNAs could target the same genes in cells.18, 20 In addition, we will investigate the effects of miR‐92b‐3p in HCC in our future work.

5. CONCLUSION

This study demonstrated that miR‐216b targeted USP28 and inhibited its expression in HCC cells. Targeting miR‐216b/USP28 axis could be as a potential strategy for HCC diagnosis and treatment in clinic.

CONFLICT OF INTEREST

The author declares no conflict of interest.

Zhang J‐F. MicroRNA‐216b suppresses the cell growth of hepatocellular carcinoma by inhibiting Ubiquitin‐specific peptidase 28 expression. Kaohsiung J Med Sci. 2020;36:423–428. 10.1002/kjm2.12193