Colocalization of MID1IP1 and c-Myc is Critically Involved in Liver Cancer Growth via Regulation of Ribosomal Protein L5 and L11 and CNOT2.

Though midline1 interacting protein 1 (MID1IP1) was known as one of the glucose-responsive genes regulated by carbohydrate response element binding protein (ChREBP), the underlying mechanisms for its oncogenic role were never explored. Thus, in the present study, the underlying molecular mechanism of MID1P1 was elucidated mainly in HepG2 and Huh7 hepatocellular carcinoma cells (HCCs). MID1IP1 was highly expressed in HepG2, Huh7, SK-Hep1, PLC/PRF5, and immortalized hepatocyte LX-2 cells more than in normal hepatocyte AML-12 cells. MID1IP1 depletion reduced the viability and the number of colonies and also increased sub G1 population and the number of TUNEL-positive cells in HepG2 and Huh7 cells. Consistently, MID1IP1 depletion attenuated pro-poly (ADP-ribose) polymerase (pro-PARP), c-Myc and activated p21, while MID1IP1 overexpression activated c-Myc and reduced p21. Furthermore, MID1IP1 depletion synergistically attenuated c-Myc stability in HepG2 and Huh7 cells. Of note, MID1IP1 depletion upregulated the expression of ribosomal protein L5 or L11, while loss of L5 or L11 rescued c-Myc in MID1IP1 depleted HepG2 and Huh7 cells. Interestingly, tissue array showed that the overexpression of MID1IP1 was colocalized with c-Myc in human HCC tissues, which was verified in HepG2 and Huh7 cells by Immunofluorescence. Notably, depletion of CCR4-NOT2 (CNOT2) with adipogenic activity enhanced the antitumor effect of MID1IP1 depletion to reduce c-Myc, procaspase 3 and pro-PARP in HepG2, Huh7 and HCT116 cells. Overall, these findings provide novel insight that MID1IP1 promotes the growth of liver cancer via colocalization with c-Myc mediated by ribosomal proteins L5 and L11 and CNOT2 as a potent oncogenic molecule.


Introduction
Though hepatocellular carcinoma (HCC) is reported to be the sixth most common cancer in the world according 2018 Global Cancer Statistics [1], more effective therapies are still being required compared to classical treatments such as chemotherapy with a kinase inhibitor sorafenib [2,3], surgery [4], immunotherapy [1] and liver transplantation [5]. Recently, multi-target therapy has been attractive in HCC treatment [5,6]. Accumulating evidence reveals that HCC progression is closely associated with epithelial-mesenchymal transition (EMT), tumor microenvironment, tumor-stromal interactions, senescence bypass and cancer stem cells [7].
Among oncogenic molecules, Myc is elevated or dysregulated in approximately 70% of human cancers, including HCC [8,9]. Indeed, the MYC oncogene family comprising of C-MYC, MYCN and MYCL encode c-Myc, N-Myc and L-Myc, which are involved in ribosome biogenesis, cell-cycle progression, protein translation and metabolism, with a variety of biological functions including proliferation, differentiation, survival and immune surveillance [10]. Also, Myc is known to regulate ribosome biogenesis and protein synthesis through the transcriptional control of RNA and protein components of ribosomes [11].
It is well documented that ribosomal RNA (rRNA) is transcribed from ribosomal DNA (rDNA) to bind to ribosomal proteins, which have the small subunit consisting of a single rRNA chain and 33 ribosomal proteins, and the large subunit including three rRNA chains and 47 ribosomal protein large subunits (RPLs) in humans [12,13]. Also, emerging evidence reveals that ribosomal protein mutations are critically involved in ribosomopathies and carcinogenesis [14], since ribosomal proteins such as L5, L11, L18 and L29 are influenced by oncogenic factors and dysregulated translational proteins [15][16][17][18].
Thus, in the present study, considering that cancer cells favor metabolism through glycolysis rather than efficient oxidative phosphorylation [25,26], the underlying oncogenic potential of MID1IP1 was explored in HCC growth in association with c-Myc signaling mediated by ribosomal protein L5 or L11 and CNOT2 in HCC cells and tissues.

Cytotoxicity Assay
HepG2 and Huh7 cells transfected with MID1IP1 siRNA or scramble siRNA control were seeded into a 96-well plate at a density of 7 × 10 3 cells/well and incubated overnight at 37 • C for 72 h. Then, 30 µL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 1 mg/mL, Merck KGaA, Darmstadt, Germany) was distributed to each well of the plate and incubated for 2 h at 37 • C in the dark. The supernatant was carefully aspirated and 100 µL of dimethyl sulfoxide (DMSO) (Ducksan, Korea) was added and the optical density values were measured in a Biorad microplate reader model 680 (Biorad, Hercules, CA, USA) at 570 nm.

Colony Formation Assay
HepG2, Huh7 and Hep3B cells transfected with MID1IP1 siRNA or scramble siRNA control were distributed onto a 12-well cell culture plate at 1 × 10 3 cells/well and incubated for 1 week to form colonies at 37 • C with 5% CO 2 . The colonies were stained with Diff Quick solution 2 (Cat No.38721, Sysmex corporation, Kobe, Hyogo, Japan), dried overnight and counted.

Cell Cycle Analysis
Based on Jung et al.'s paper [27], briefly, HepG2 and Huh7 cells transfected with MID1IP1 siRNA or scramble siRNA control were cultured to confluency for 72 h and then were harvested by using a Becton Dickinson (BD) falcon cell scraper. The detached cells were washed twice with cold PBS and fixed in 75% ethanol at −20 • C for overnight. The cells were incubated with RNase A (10 mg/mL) for 1 h at 37 • C and then stained with propidium iodide (50 µg/mL) for 30 min. The stained cells were analyzed with FACSCalibur by using CellQuest Software version 5.2.1 (Becton Dickinson, Franklin Lakes, NJ, USA).

Terminal Deoxynucleotidyl Transferase-dT-Mediated dUTP Nick End Labelling (TUNEL) Assay
As shown in Jung et al.'s paper [28], HepG2 and Huh7 cells transfected with MID1IP1 siRNA or scramble siRNA control were cultured for 72 h, fixed in permeabilization solution and incubated with TUNEL assay mixture for 60 min. The TUNEL-stained cells were observed under a FLUOVIEW FV10i confocal microscope (Olympus, Tokyo, Japan).

c-Myc Stability Assay Using Cycloheximide
Based on Lee et al.'s paper [29], HepG2 and Huh7 cells transfected with MID1IP1 siRNA or negative control siRNA were cultured with 50 µg/mL cycloheximide for various times (0, 15 and 60 min) and were subjected to Western blotting with antibodies of c-Myc, MID1IP1, β actin and/or snail, p21.

Immunofluorescence Assay
HepG2 and Huh7 cells transfected with MID1IP1 siRNA or negative control siRNA were fixed with 4% formaldehyde and were permeabilized in 0.1% Triton X-100, based on Lee et al.'s paper [29]. The fixed cells were washed and incubated with the specific antibodies of MID1IP1 (1:200; Invitrogen, Waltham, MA, USA) and c-Myc (1:500; Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 • C and then with secondary antibodies of Alex Fluor 488 goat rabbit immunoglobin G (IgG) antibody (1:500) (Invitrogen, Waltham, MA, USA) and Alexa Fluor 546 goat mouse-IgG antibody (1:500) (Invitrogen, Waltham, MA, USA) for 2 h at room temperature. The nuclei of the cells were stained with 4,6-diamidino-2-phenylindole (DAPI) and then were observed by using a FLUOVIEW FV10i confocal microscope and also the images of MID1IP1 and c-Myc-stained cells were taken by a Delta Vision imaging system.

Statistical Analysis
All data were expressed as means ± standard deviation (SD). Student's t-test was used for comparison of two groups. Also, the one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test was performed for multi-group comparison using GraphPad Prism software (Version 5.0, San Diego, CA, USA). For overall survival (OS), associations between MID1IP1 subtypes and survival rate were then calculated by Kaplan-Meier analysis using a log-rank test.

Endogenous Expression Levels in a Variety of Cells and Effect of MID1IP1 Depletion on the Viability and the Number of Colonies in HCCs
To assess the effect of MID1IP1 depletion on the viability and proliferation of HCCs, MTT assay and colony formation assay were conducted in HCCs. MID1IP1 was highly expressed in HepG2, Huh7, SK-Hep1, PLC/PRF5 and immortalized hepatocyte LX-2 cells more than in normal hepatocyte AML-12 cells and Hep3B cells ( Figure 1A). Also, depletion of MID1IP1 depletion reduced the viability of Huh7 and Hep3B cells in a time-dependent fashion compared to the untreated control ( Figure 1B). Also, the number of colonies were significantly reduced in MID1IP1-depleted HepG2, Huh7 and Hep3B cells compared to the untreated control ( Figure 1C).

MID1IP1 Depletion Increases Sub G1 Population and Increases the Number of TUNEL-Positive Cells in HepG2 and Huh7 Cells
To check whether the cytotoxicity by MID1IP1 depletion is due to apoptosis, cell cycle analysis and TUNEL assay were performed in HepG2 and Huh7 cells. Here, MID1IP1 depletion increased the sub G1 population (Figure 2A) and the number of TUNEL-positive cells ( Figure 2B) in HepG2 and Huh7 cells compared to the untreated control.

MID1IP1 Depletion Increases Sub G1 Population and Increases the Number of TUNEL-Positive Cells in HepG2 and Huh7 Cells
To check whether the cytotoxicity by MID1IP1 depletion is due to apoptosis, cell cycle analysis and TUNEL assay were performed in HepG2 and Huh7 cells. Here, MID1IP1 depletion increased the sub G1 population (Figure 2A) and the number of TUNEL-positive cells ( Figure 2B) in HepG2 and Huh7 cells compared to the untreated control. siRNA plasmid or negative control siRNA were cultured for 72 h and then were fixed with 4% paraformaldehyde for 30 min, exposed to TUNEL assay mixture for 60 min and TUNEL-stained cells were visualized by a FLUOVIEW FV10i confocal microscopy (Olympus, Tokyo, Japan).

MID1IP1 Depletion Attenuates the Expression of Pro-PARP, c-Myc and CNOT2 and Activates p21 in Cancers
Consistently, to confirm the effect of MID1IP1 depletion on apoptosis-related proteins, Western blotting was conducted in HCCs and HCT116 cells. Here, MID1IP1 depletion attenuated the expression of pro-PARP, MID1IP1 and c-Myc in HepG2 and Huh7 cells compared to the untreated control ( Figure 3A). In contrast, MID1IP1 overexpression activated c-Myc and reduced p21 in HepG2 cells ( Figure 3B). Also, MID1IP1 depletion cleaved PARP in Hep3B cells ( Figure 3C). Similarly, MID1IP1 depletion attenuated the expression of c-Myc, pro-PARP and CNOT2 in HCT116 cells in a concentration-dependent fashion ( Figure 3D). HepG2 and Huh7 cells transfected with MID1IP1 siRNA plasmid or negative control siRNA were cultured for 72 h and then were fixed with 4% paraformaldehyde for 30 min, exposed to TUNEL assay mixture for 60 min and TUNEL-stained cells were visualized by a FLUOVIEW FV10i confocal microscopy (Olympus, Tokyo, Japan).

MID1IP1 Depletion Attenuates the Expression of Pro-PARP, c-Myc and CNOT2 and Activates p21 in Cancers
Consistently, to confirm the effect of MID1IP1 depletion on apoptosis-related proteins, Western blotting was conducted in HCCs and HCT116 cells. Here, MID1IP1 depletion attenuated the expression of pro-PARP, MID1IP1 and c-Myc in HepG2 and Huh7 cells compared to the untreated control ( Figure 3A). In contrast, MID1IP1 overexpression activated c-Myc and reduced p21 in HepG2 cells ( Figure 3B). Also, MID1IP1 depletion cleaved PARP in Hep3B cells ( Figure 3C). Similarly, MID1IP1 depletion attenuated the expression of c-Myc, pro-PARP and CNOT2 in HCT116 cells in a concentration-dependent fashion ( Figure 3D).

MID1IP1 Depletion Attenuates c-Myc Stability in HepG2 and Huh7 Cells in the Presence of Cycloheximide
To confirm the effect of MID1IP1 depletion on c-Myc stability, DNA synthesis inhibitor cycloheximide assay was performed in HepG2 and Huh7 cells. Herein, MID1IP1 depletion synergistically suppressed c-Myc stability compared to the cycloheximide-alone control in Huh7 cells ( Figure 4A). Likewise, MID1IP1 depletion synergistically reduced the expression of c-Myc, p21 and snail compared to the cycloheximide-alone control in HepG2 cells ( Figure 4B).

MID1IP1 Depletion Attenuates c-Myc Stability in HepG2 and Huh7 Cells in the Presence of Cycloheximide
To confirm the effect of MID1IP1 depletion on c-Myc stability, DNA synthesis inhibitor cycloheximide assay was performed in HepG2 and Huh7 cells. Herein, MID1IP1 depletion synergistically suppressed c-Myc stability compared to the cycloheximide-alone control in Huh7 cells ( Figure 4A). Likewise, MID1IP1 depletion synergistically reduced the expression of c-Myc, p21 and snail compared to the cycloheximide-alone control in HepG2 cells ( Figure 4B).

Colocalization Between MID1IP1 and c-Myc in Human HCC Tissues, HepG2 and Huh7 Cells
It is well known that colocalization indicates the spatial overlap between two or more different fluorescent targets located in the same area of the cell [30]. Here, human HCC patient tissue microarray strongly demonstrated the close association between MID1IP1 and c-Myc, with a significant correlation coefficient (p = 0.004) by Fisher's exact test, since MID1IP1 and c-Myc were coexpressed in the same area of HCC tissue ( Figure 5A). Consistently, Immuoflourescence showed colocalization between MID1IP1 and c-Myc with a yellowish color when merging the fluorescence of green (MID1IP1) and red (c-Myc) ( Figure 5B).

Colocalization Between MID1IP1 and c-Myc in Human HCC Tissues, HepG2 and Huh7 Cells
It is well known that colocalization indicates the spatial overlap between two or more different fluorescent targets located in the same area of the cell [30]. Here, human HCC patient tissue microarray strongly demonstrated the close association between MID1IP1 and c-Myc, with a significant correlation coefficient (p = 0.004) by Fisher's exact test, since MID1IP1 and c-Myc were coexpressed in the same area of HCC tissue ( Figure 5A). Consistently, Immuoflourescence showed colocalization between MID1IP1 and c-Myc with a yellowish color when merging the fluorescence of green (MID1IP1) and red (c-Myc) ( Figure 5B).

Colocalization Between MID1IP1 and c-Myc in Human HCC Tissues, HepG2 and Huh7 Cells
It is well known that colocalization indicates the spatial overlap between two or more different fluorescent targets located in the same area of the cell [30]. Here, human HCC patient tissue microarray strongly demonstrated the close association between MID1IP1 and c-Myc, with a significant correlation coefficient (p = 0.004) by Fisher's exact test, since MID1IP1 and c-Myc were coexpressed in the same area of HCC tissue ( Figure 5A). Consistently, Immuoflourescence showed colocalization between MID1IP1 and c-Myc with a yellowish color when merging the fluorescence of green (MID1IP1) and red (c-Myc) ( Figure 5B).

The Critical Role of Ribosomal Protein L5 or L11 in c-Myc Inhibition by MID1IP1 Depletion in HepG2 and Huh7 Cells
Given that ribosomal proteins such as L5, L11, L18 and L29 are known to be influenced by oncogenic factors and dysregulated translational proteins [15], the critical role of ribosomal protein L5 or L11 was explored in c-Myc inhibition by MID1IP1 depletion in HepG2 and Huh7 cells. Here, MID1IP1 depletion activated L5 and L11 and reduced c-Myc, while loss of L5 or L11 reversed the inhibitory effect of MID1IP1 depletion on c-Myc in HepG2 ( Figure 6A,B) and Huh7 cells ( Figure  6C,D).

The Critical Role of Ribosomal Protein L5 or L11 in c-Myc Inhibition by MID1IP1 Depletion in HepG2 and Huh7 Cells
Given that ribosomal proteins such as L5, L11, L18 and L29 are known to be influenced by oncogenic factors and dysregulated translational proteins [15], the critical role of ribosomal protein L5 or L11 was explored in c-Myc inhibition by MID1IP1 depletion in HepG2 and Huh7 cells. Here, MID1IP1 depletion activated L5 and L11 and reduced c-Myc, while loss of L5 or L11 reversed the inhibitory effect of MID1IP1 depletion on c-Myc in HepG2 ( Figure 6A,B) and Huh7 cells ( Figure 6C,D).

CNOT2 Knockdown Enhances Antitumor Effect of MID1IP1 Depletion in HepG2, Huh7 and HCT116 Cells
To assess the important role of CNOT2 in MIDIIP1-mediated oncogenesis in HCCs, a CNOT2 siRNA study was conducted in HepG2 and Huh7 cells, since several oncogenic factors are orchestrated in carcinogenesis [31]. Here, CNOT2 knockdown enhanced the inhibitory effect of MID1IP1 depletion on c-Myc, pro-PARP and pro-casapse 3 in HepG2 cells, and also CNOT2 depletion promoted the inhibitory effect of MID1IP1 depletion on c-Myc, pro-PARP and pro-casapse3 in Huh7 cells ( Figure 7A,B). Furthermore, CNOT2 knockdown enhanced the inhibitory effect of MID1IP1 depletion on c-Myc and pro-PARP, even in HCT116 colorectal cancer cells ( Figure 7C).

CNOT2 Knockdown Enhances Antitumor Effect of MID1IP1 Depletion in HepG2, Huh7 and HCT116 Cells
To assess the important role of CNOT2 in MIDIIP1-mediated oncogenesis in HCCs, a CNOT2 siRNA study was conducted in HepG2 and Huh7 cells, since several oncogenic factors are orchestrated in carcinogenesis [31]. Here, CNOT2 knockdown enhanced the inhibitory effect of MID1IP1 depletion on c-Myc, pro-PARP and pro-casapse 3 in HepG2 cells, and also CNOT2 depletion promoted the inhibitory effect of MID1IP1 depletion on c-Myc, pro-PARP and pro-casapse3 in Huh7 cells ( Figure 7A,B). Furthermore, CNOT2 knockdown enhanced the inhibitory effect of MID1IP1 depletion on c-Myc and pro-PARP, even in HCT116 colorectal cancer cells ( Figure 7C).

Discussion
In the current work, the underlying molecular mechanism of MID1IP1 as a potent oncogene was elucidated in association with c-Myc mediated by ribosomal protein L5 or L11 and CNOT2 in HCCs, with the hypothesis that MID1IP1 with hyperlipidemic activity [19,20] may act as a potent oncogene in HCC progression. Herein, MID1IP1 was highly expressed in HepG2, Huh7, SK-Hep1, PLC/PRF5 and immortalized hepatocyte LX-2 cells more than in normal hepatocyte AML-12 cells and Hep3B cells, indicating endogenous overexpression of MID1IP1 in HCCs, except for p53-deficient Hep3B cells.
In the present study, MID1IP1 depletion showed cytotoxic and anti-proliferative effects and increased the sub G1 population and the number of TUNEL-positive cells in HepG2 and Huh7 cells, indicating that MID1IP1 is involved in the growth, proliferation and antiapoptosis of HCCs.
Accumulating evidence reveals that overabundance and mutations of the Myc family comprising of c-Myc, N-Myc and L-Myc are shown in several cancers [32]. Among many apoptosisrelated proteins, proteolytic cleavages of PARP into 89 kDa and 24 kDa fragments by caspases are considered an early indicator of apoptosis [33] and caspase 3 is known as an executioner caspase in apoptosis by the destruction of cellular structures ,including DNA fragmentation or cytoskeletal proteins [34]. p21, also termed p21 WAF1/Cip1 , is known as a key cell cycle regulator that arrests cells at the G1 and G2 phases and is also reported to trigger apoptosis [35]. Our Western blotting revealed that MID1IP1 depletion attenuated pro-PARP, c-Myc and activated p21 in HepG2 and Huh7 cells, while MID1IP1 overexpression activated c-Myc and reduced p21 in HepG2 cells, demonstrating the

Discussion
In the current work, the underlying molecular mechanism of MID1IP1 as a potent oncogene was elucidated in association with c-Myc mediated by ribosomal protein L5 or L11 and CNOT2 in HCCs, with the hypothesis that MID1IP1 with hyperlipidemic activity [19,20] may act as a potent oncogene in HCC progression. Herein, MID1IP1 was highly expressed in HepG2, Huh7, SK-Hep1, PLC/PRF5 and immortalized hepatocyte LX-2 cells more than in normal hepatocyte AML-12 cells and Hep3B cells, indicating endogenous overexpression of MID1IP1 in HCCs, except for p53-deficient Hep3B cells.
In the present study, MID1IP1 depletion showed cytotoxic and anti-proliferative effects and increased the sub G1 population and the number of TUNEL-positive cells in HepG2 and Huh7 cells, indicating that MID1IP1 is involved in the growth, proliferation and antiapoptosis of HCCs.
Accumulating evidence reveals that overabundance and mutations of the Myc family comprising of c-Myc, N-Myc and L-Myc are shown in several cancers [32]. Among many apoptosis-related proteins, proteolytic cleavages of PARP into 89 kDa and 24 kDa fragments by caspases are considered an early indicator of apoptosis [33] and caspase 3 is known as an executioner caspase in apoptosis by the destruction of cellular structures, including DNA fragmentation or cytoskeletal proteins [34]. p21, also termed p21 WAF1/Cip1 , is known as a key cell cycle regulator that arrests cells at the G1 and G2 phases and is also reported to trigger apoptosis [35]. Our Western blotting revealed that MID1IP1 depletion attenuated pro-PARP, c-Myc and activated p21 in HepG2 and Huh7 cells, while MID1IP1 overexpression activated c-Myc and reduced p21 in HepG2 cells, demonstrating the pivotal role of MID1IP1 in HCC progression. Furthermore, MID1IP1 depletion enhanced suppression of c-Myc stability in HepG2 and Huh7 cells in the presence of DNA synthesis inhibitor cycloheximide, implying that MID1IP1 regulates c-Myc stability.
It is well documented that Myc acts as a regulator of ribosome biogenesis and protein synthesis [11,36] and the ribosomal large subunit comprises three rRNA chains and 47 RPLs in humans [37].
Here, MID1IP1 depletion induced upregulation of ribosomal protein L5 or L11, whereas depletion of L5 or L11 activated c-Myc in MID1IP1-depleted HepG2 and Huh7 cells, implying that L5 or L11 as a tumor suppressor regulates c-Myc, which was supported by Liao et al.'s paper that ribosomal proteins L5 and L11 cooperatively inactivate c-Myc via RNA-induced silencing complex [17]. Consistently, human tissue array showed that the overexpression of MID1IP1 was colocalized with c-Myc in human HCC tissues, with a significant correlation coefficient by Fisher's exact test and also, Immunofluorescence confirmed the yellowish color by merging green and red signals between MID1IP1 and c-Myc in HepG2 and Huh7 cells, strongly demonstrating colocalization between MID1IP1 and c-Myc.
Emerging evidence reveals that CNOT2 acts as an oncogene [22,23], adipogenic molecule [21] and autophagy regulator [22,38]. Considering that nonalcoholic fatty liver disease (NAFLD) increases the risk of HCC progression [39], and obesity-like condition promotes the proliferation of breast cancer via Warburg effect inversion [40], hyperlipidemic or adipogenic molecules may be similarly involved in cancer progression. Indeed, CNOT2 depletion enhanced the antitumor effect of MID1IP1 depletion to reduce c-Myc, procaspase 3 and pro-PARP in MID1IP1-depleted HepG2, Huh7 and HCT116 cells, suggesting a close association between MID1IP and CNOT2. However, further study is required to evaluate the pivotal roles and protein-protein interaction (PPI) of MID1IP1 and CNOT2 in cancers via the Warburg effect in vitro and in vivo.
Taken together, these findings provide novel insight that MID1IP1 promotes the growth of liver cancer via colocalization with c-Myc mediated by ribosomal protein L5 and L11 and CNOT2 as a potent oncogenic molecule.

Conclusions
Collectively, through our current study, MID1IP1 depletion reduced the viability and proliferation, and increased the sub G1 population and the number of TUNEL-positive cells in HepG2 and Huh7 cells. Consistently, MID1IP1 depletion attenuated pro-PARP, c-Myc and activated p21 and synergistically attenuated c-Myc stability by cycloheximide assay in HepG2 and Huh7 cells, while MID1IP1 overexpression activated c-Myc and reduced p21 in HepG2 cells. Notably, MID1IP1 depletion upregulated L5 or L11, while loss of L5 or L11 rescued c-Myc in MID1IP1-depleted HepG2 and Huh7 cells. Interestingly, MID1IP1 was colocalized with c-Myc in human HCC cells and human tissues by IHC and Immunofluorescence. Furthermore, CNOT2 depletion enhanced the antitumor effect of MID1IP1 depletion to reduce c-Myc, procaspase 3 and pro-PARP in MID1IP1-depleted HepG2, Huh7 and HCT116 cells. Overall, these findings suggest that MID1IP1 promotes liver cancer progression via colocalization with c-Myc mediated by ribosomal protein L5 and L11 and CNOT2 as a potent oncogenic molecule.