KRT23 acts as an oncogene in hepatocellular carcinoma by regulating PI3K/AKT/GSK3β pathway via P21

doi:10.21203/rs.3.rs-4721250/v1

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KRT23 acts as an oncogene in hepatocellular carcinoma by regulating PI3K/AKT/GSK3β pathway via P21

https://doi.org/10.21203/rs.3.rs-4721250/v1

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Hepatocellular carcinoma (HCC) is a leading cancer worldwide. Keratin23 maybe a potential biomarker for HCC development; however, regulatory mechanisms remain unclear. In this research we explored the expression and effect of KRT23 in HCC. GEPIA, Kaplan-Merier survival analysis IHC, qRT-PCR and western blot were applied to further detect the expression of KRT23, as well as prognosis. Functionally, Relative cell biology experiments were applied. Moreover, xenograft tumors were conducted in vivo. Mechanically, Immunofluorescence, western blot and Co-immunoprecipitation were operated. As for results, we discovered the high expression of KRT23 in HCC. Functionally, KRT23 knockdown reduced cell proliferation and metastasis. KRT23 knockdown inhibited EMT and PI3K/AKT/GSK3β pathway. In summary, KRT23 accelerated HCC proliferation and metastasis by regulating PI3K/AKT/GSK3β pathway via P21.

Biological sciences/Cancer/Metastases

Biological sciences/Cancer/Oncogenes

Biological sciences/Cancer/Tumour biomarkers

keratin 23

hepatocellular carcinoma

epithelial mesenchymal transition

PI3K/AKT/GSK3β pathway

P21

Hepatocellular carcinoma (HCC) is still a noteworthy public health issue all over the world even in the coming decade in wake of its fast - rising incidence and high mortality[1, 2]. As we all known, HCC development results from gradual accumulation of multiple genetic and factorial regulation, including hepatitis B and C viruses, alcohol consumption, aflatoxin exposure, obesity and some with no specific reason[3, 4]. Detection and treatments for HCC has been advanced greatly over the past few years, but there is no promising improvement for patients, along with 5 -year survival rate lower than 18% caused by rather high diagnosis at advanced stage and then limited treatment options[5–7]. Considering the correlation between early detection and prognosis for patients, it’s urgent to discover new biomarkers for HCC patients at risk and much more sensitive targets for current therapies[8].

Expressed in a tissue-specific manner, keratins (KRTs) are accepted as structural proteins that has the intermediate filaments of epithelial cells[9]. Keratin family possesses 50 members, in which KRT9-23 are classified to the acidic type while KRT1-8 are basic-neural[10]. Interacted with other proteins or other members in KRT family with various epigenetic modification, keratins take great effect on stress response, several cancers progression and signaling pathways[11]. As a member of acidic keratins, KRT23 was firstly discovered in pancreatic cancer cells and was induced by histone hyperacetylation[12]. Meanwhile, the high expression of KRT23 in microsatellite-stable colon cancer (MSS) was applied for distinction MSS from microsatellite-instable colon cancers (MSI)[10]. Moreover, KRT23 as a stress-inducible marker, correlated with the liver disease severity[9], which predicted that KRT23 might participate in liver disease even cancer. All above reminds us the orientation of KRT23 on HCC development.

For this research, bioinformatic analyses were firstly applied and revealed the high expression of KRT23 in HCC patients and correlation of poor prognosis. Next functional experiments in vitro and xenograft tumors in nude mice were conducted to detect the effect of KRT23 in HCC progression. Furthermore, we found that KRT23 also mediated EMT and interacted with P21 to regulate PI3K/AKT/GSK3β pathway. Above all, KRT23 may has the potential as a new drug target and diagnostic biomarker for HCC patients.

Bioinformatic analysis

Gene Expression Profiling Interactive Analysis (GEPIA) was applied for detecting the expression of KRT23 in HCC and normal patients, including 369 liver tumors and 160 normal tissues from the TCGA and the GTEx projects[13]. Then Kaplan-Merier Plotter database was used for the prediction of patients’ prognosis[14]. Meanwhile the interaction between KRT23 and other proteins was explored in STRING 10.5[15, 16], which is a well-known database for predicting protein-protein interactions.

Cell culture and tumor tissues collection

All cells involved in this research were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were Cultured with DMEM of high glucose (Hyclone, America), which contained 10% fetal bovine serum (FBS, Gibico, America) and 100 U/ml penicillin-streptomycin (Geneview, America). Meanwhile all cells were maintained in sterile and humid cell incubator with 5% CO₂ at 37℃ and stored at liquid nitrogen. On the other hand, clinical tumor samples and tumor adjacent samples around tumors 5 cm were collected from pathology department of First Affiliated Hospital of Xi'an Jiaotong University after approved and authorized by the institutional committee of medical ethics, which means tissues involved all had gotten informed consent from patients. We promise that all methods involved were performed in accordance with the relevant guidelines and regulations.

Plasmid construction and Cell transfection assay

Specific interfering RNAs against the sequence of KRT23 or P21 were all designed and synthesized by Genepharma (Shanghai, China) and concrete sequences has been listed in Supplementary table 1. In view of transfection efficiency, siKRT23#1 was selected for further experiment and construction of lentivirus which inserting the sequence of siKRT23#1 into the vector GV248. Lentivirus-KRT23-RNAi was bought from GeneChem (Shanghai, China) and cells transfected with lentivirus were cultured with DMEM/high glucose containing 5 µg/ml puromycin. For overexpression, KRT23 sequence was inserted into GV219 vector for cloning, which was also designed and constrcted by GeneChem (Shanghai, China). Lipofectamine2000 Transfection Reagent (Geneview) was applied as its instruction for importing siRNAs or plasmid into cells when the cell density attached around 50%-70%.

RNA isolation and quantitative real-time PCR (qRT-PCR)

RNA extraction was conducted as the protocol of Trizol Reagent (Invitrogen, USA). The sequences of primers applied for this research had been displayed in Supplementary table 2. cDNAs were synthesized as the instruction of EasyQuick RT MasterMix (CWBIO, China) and qRT-PCR was examined using UltraSYBR Mixture ((CWBIO, China). β-actin was regarded as the control reference when the expression of genes was calculated as the formula 2^−ΔΔCt.

MTT assay

MTT assay was used to analyze the cell proliferation after transfection. Cells were seeded into 96-well plates at a density of 5000 cells/well. In the following days, 10 µL of 5 mg/ml MTT was added into every well and after incubating for 4 h in cell incubator at 37℃, discarded the suspension and 150 µL DMSO were applied for purple crystal solution at the bottom of 96-well plates. At the end, EnSpire Multimode Plate Reader (PerkinElmer) was used for measuring the optical density (OD) at 490nm.

Colony formation assay

Stable transfected cells were seeded into 6-well plates for 500 cells/well. When one separate cell cloned and formed clusters along with no less than 50 cells after 14 days, the colony formation was fixed by methanol for 15 min and then colored with 0.1% crystal violet for 20 min. Images were collected under microscope when the plates were dry.

Flow cytometry

Cell apoptosis was conducted as the instruction of Annexin V-APC/7-AAD apoptosis kit (MULTI SCIENCES, China). After transfection for 48 h, cells were gathered and washed by PBS for two times. Then 500 µL 1×Binding buffer was applied to resuspend cells and following 5 µL Annexin V-APC and 10 µL 7-AAD were added for incubation away from light at room temperature for 5 min. Finally, samples were examined by FALS CALIBAR (USA). As for cell cycle, as the protocol of Cell Cycle Detection Kit (KeyGEN BioTECH, China), 1×10⁶ cells were collected and washed by PBS after transfection for 48 h. Then 70% cold ethyl alcohol was supplied for fixing cells overnight at 4℃. The next day, PBS was used to wipe off the fixing liquid and then 500 µL PI/RNase A solution prepared in advance at adequate ratio of 9:1 were added and incubated at room temperature for 60 min. identically, samples were detected on FALS CALIBAR (USA).

Transwell assay

Chambers of 8µm pole size coated with Matrigel (BD Biosciences) were applied for cell invasion. After transfection for 24 h, cells were resuspended using medium with no FBS and seeded on the top of chambers which had been put into 600 µL medium with 10% FBS in 24-well plates. Chambers were taken out after placed in cell incubator for 24 h and washed for 3 times with PBS. Wiped gently with swabs internally, chambers were put into 95% alcohol for fixation for 15 min and then stained by 0.1% crystal violet for 20 min. Images were collected after removing superfluous crystal violet by PBS under the microscope at a magnification of 200×. Considering migration, the protocol was similar with that for invasion, just with no Matrigel for chambers.

Wound healing experiment

Stable transfected cells were seeded into 6-well plates to attach a confluence of 90%-95% and after stable adhesion, 10 µL sterile tips were applied for scratching. In case of interference from cell growth, medium with no FBS was used during the period. Pictures were collected respectively at 0 h, 24 h, and 48 h under the microscope at an amplification of 200×.

The oncogenicity in nude mice

Randomly divided into 2 groups, male athymic 4-week-old nude mice were purchased from the Centre Animal laboratory of Xi’an Jiaotong university and their good care was strictly according to the institution guidelines. Stable- transfected cells were used for xenograft tumors. 200 µL stably transfected cells HepG2 containing at least 5×10⁶ cells in PBS were injected subcutaneously for detection of cell growth in vivo. Tumor volumes were measured every four days for drawing the growth curve over time. Mice were were narcotized by using ketamine for 100mg/kg and sacrificed using cervical dislocation method. Xenograft tumors were collected after 32 days. As for metastasis, 100 µL stably transfected cells HepG2 were injected slowly from tail vein and after 2 months mice involved were narcotized using similar method above. Picking one eyeball swiftly and when blood was almost over, lung tissues were taken out for research. H&E staining was conducted to assess pulmonary metastasis. The animal experiment was thoroughly operated according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and was authorized by the medical ethics committee of the First Affiliated Hospital of Xi'an Jiaotong University.

H&E staining and Immunohistochemistry (IHC)

Xenograft tumors and clinical tissue samples were stored at 10% formalin overnight and then embedded by paraffin. All conduction was operated as the protocol of Kit of Streptomyces Habilis Ovalbumin – Biotin (ZSGB-BIO, China). Thickness for 4mm and put on slides, those tissues was dewaxed using xylene and then hydrated by grade-reduced concentration of alcohol. Then stained with H&E and observed using microscope. As for IHC, primary antibodies were incubated overnight at 4℃ after dewaxing and hydration. The next day secondary antibodies were incubated and then detected using DAB solution. Stained with hematoxylin for cell nucleus, next slides were dehydrated by grade-ascending concentration of alcohol and then transparentized by xylene. Finally sealed by coverslips with neural gum, the slides were observed by microimaging scanning system (Leica). During this process, the blocking time for KRT23 primary antibody and secondary antibody, as well as the exposure time for DAB staining were strictly controlled consistency. Meanwhile relative parameter settings for slide scanner were always constant. Antibodies applied in detail has been listed in Supplementary table 3.

CO-immunoprecipitation (CO-IP) and western blot

RIPA buffer with proteinase and phosphatase inhibitors was applied for cell protein isolation on ice for 10 min and then primary antibodies or IgG were added for incubation at room temperature for 4 h. Next, activated beads were mixed with protein lysates above and kept shocking at 4℃ overnight. The next day, beads were washed for 3 times using RIPA buffer and resuspended by 1× loading buffer. Conduction remaining was similar with western blot. Proteins were uploaded into 5% gel for concentration and then 10% for dividing during electrophoresis process. Following proteins were transferred onto PVDF membrane with 0.45 µm or 0.22 µm pole size, which were blocked with 5% milk without fat and then primary antibodies were incubated respectively at 4℃ overnight. Secondary antibodies were incubated for 1 h at room temperature the next day. All bands were observed by FD^TMFDbio- Dura Ecl kit (Fdbio science, China). Application for antibodies in detail was shown in Supplementary table 3.

Immunofluorescence

Sterile slides were put into 6-well plates before stable transfected cells were seeded into plates. Slides with adherent cells were fixed with 4% paraformaldehyde for 15 min and then blocked with goat serum for 30 min. Following primary antibodies were incubated overnight at 4℃ in a wet black box. The next day, after washed for 3 times by PBS, slides were incubated with secondary antibodies for 1 h at room temperature. After stained with DAPI for nucleus and covered with anti-fading buffer, images were collected under inverted immunofluorescent microscope (Leica). The details for antibodies showed in Supplementary table 3.

Statistical analysis

The software SPSS 23.0 was applied for statistical analysis. Difference between two paired groups were analyzed by Student’s t-test and χ2 test was applied for analysis of clinical data. p value < 0.05 was considered as significant difference and p < 0.01 very significant.

Overexpression of KRT23 in HCC samples and HCC cell lines

As is shown in Fig. 1A, the results of bioinformatic analysis from GEPIA revealed that the expression of KRT23 was higher when compared with that in normal patients, 369 liver tumors and 160 normal tissues from the TCGA and the GTEx projects involved (*p<0.01). Meanwhile, split by median cutoff, the overexpression of KRT23 indicated poor prognosis that the overall survival for HCC patients with higher expression of KRT23 was shorter than those with lower expression (Fig. 1B, p=0.038). Next, the results of immunochemistry (IHC) showed that under the same condition, the staining intensity for KRT23 in tumor group was higher than that in tumor-adjacent samples, which indicated the higher expression of KRT23 in HCC tumor samples (Fig. 1C), accompanied with correlation between the expression of KRT23 and some clinical factors, as shown in Table 1, including tumor size (p=0.006), HBV infection (p=0.030), AFP (p=0.001), pathological grade (p=0.027), TNM stage (p=0.040) and cancer embolus (p=0.034). Then we detected the expression of KRT23 in HCC cell lines. The result of qRT-PCR displayed that KRT23 was overexpressed on mRNA level when compared with that in immortal hepatocyte LO2 (Fig. 1D, *p<0.05, **p<0.01), which was familiar with the result of western blot that detecting the expression of KRT23 on protein level (Fig. 1E). Moreover, immunofluorescence was applied to acknowledge the location of KRT23 in cytoplasm (Fig. 1F). In view of that, three special interfering RNAs against the sequence of KRT23 was designed and transfected effect was examined by both qRT-PCR and western blot to choose the siKRT23#1 for the research in depth (Fig. 1G and H, **p<0.01). In all, the expression of KRT23 was higher in both HCC patient samples and HCC cell lines. relating to poor prognosis.

KRT23 knockdown inhibited HCC cell proliferation, including apoptosis and cell cycle

Colony formation assay and MTT assay were conducted to assess the influence for proliferation. In colony formation assay, the number of colony formation in KRT23 knockdown group was obviously less than that in control and NC group (Fig. 2A, *p<0.05, **p<0.01). As for MTT assay, when the expression of KRT23 was knocked down, the HCC cell growth over time was retarded when compared with both control and NC group (Fig. 2B and C, *p<0.05, **p<0.01). Furthermore, flow cytometry suggested that the number of apoptotic cells in KRT23 knockdown group was increased in contrast to control and NC group (Fig. 2D and E, *p<0.05). In addition, KRT23 knockdown decreased the cell number in S phase when compared with that in control and NC group (Fig. 2F-H, *p<0.05). Following the classical biomarkers for apoptosis and cell cycle were detected by western blot (Fig. 2I and J). Just as the results shown, the expression of PARP/cleaved PARP and Bax were augmented when KRT23 was knocked down while Bcl2 was alleviated; the relative markers for cell cycle involving cyclinD1, cyclin E, CDK4, CDK6, C-MYC were all weakened in siKRT23 group. Above all, KRT23 knockdown inhibited HCC cell proliferation and accelerated apoptosis.

KRT23 knockdown repressed HCC cell migration and invasion

For this part, transwell assay and wound healing assay were used for HCC cell metastasis. In siKRT23 group, the HCC cell number for migration and invasion was apparently decreased compared with that in control and NC group (Fig. 3A and B, **p<0.01). similarly, when KRT23 was knocked down, the motility of HCC cells over time was attenuated (Fig. 3C, D and E, *p<0.05, **p<0.01). MMP9, also known as gelatinase B, which was reported could participate in the process of degradation of extracellular matrix, is responsible for metastasis and invasion of HCC cells even epithelial-mesenchymal transition in HCC[17, 18]. results above displayed that KRT23 regulated HCC cell migration and invasion, and then expression of MMP9 was detected using western blot when KRT23 was downregulated in HCC cells. As shown, KRT23 knockdown strongly abrogated its expression on protein level (Fig. 3F). To sum up, KRT23 knockdown suppressed the metastasis of HCC cells.

Upregulated KRT23 accelerated HCC cells proliferation and metastasis

Relative functional experiments were conducted after KRT23 was overexpressed by transfected with KRT23 plasmid. On the basis of expression of KRT23 in HCC cells, MHCC-97H and SMMC-7721 were selected for research of overexpression. After 48 h of transfection for KRT23 plasmid, qRT-PCR was applied for detecting the expression of KRT23 and as shown in supplementary figure 1A (*p<0.05, **p<0.01), the expression of KRT23 was elevated. Then MTT assay and colony formation were operated for assessing cell proliferation. Compared with vector group, the cell growth rate in KRT23 group was apparently improved over time (supplementary figure 1B and C, **p<0.01). Similarly, colony formation numbers in KRT23 group were much more than that in vector group (supplementary figure 1D). Following transwell assay and wound healing assay were used for metastasis. As shown in supplementary figure 1E and F (*p<0.05, **p<0.01), the number of migrated and invaded cells in KRT23 group was greatly increased when in contrast to vector group. For scratch test, KRT23 overexpression expedited the motility of HCC cells when compared with that in vector group (supplementary figure 1G-I, *p<0.05, **p<0.01). In summary, KRT23 overexpression contributed to the growth and metastasis of HCC cells.

KRT23 controlled HCC proliferation and metastasis in vivo

The function of KRT23 were further verified in vivo. For assurance, the efficiency for stably transfected cells Hep3B and HepG2 were evaluated using both qRT-PCR and western blot to be available as shown in Fig. 4A (**p<0.01). 200μl HepG2 stably transfected with sh-NC or sh-KRT23 in PBS, which contained 5×10⁶ cells was subcutaneously injected to assess tumor growth. As the image shown, the tumor volume in sh-KRT23 group was evidently less than sh-NC group (Fig. 4B), and the growth rate over time was sharply retarded when KRT23 was knocked down (Fig. 4C, **p<0.01). Considering lung metastasis models, 100 μl suspension of HepG2 stably transfected with sh-NC or sh-KRT23 was injected from tail vein. There were much more pulmonary metastatic nodules in sh-NC group when compared with that in sh-KRT23 group in which it seems hyperplasia for 400×(Fig. 4D). Moreover, the expression of Ki67 was detected and as shown in Fig. 4E and F (**p<0.01), the number of Ki67 positive cells was predominantly richer than that in sh-KRT23 group. In a word, KRT23 regulated HCC growth and pulmonary metastasis in vivo.

KRT23 regulated Epithelial-mesenchymal transition and PI3K/AKT/GSK3β pathway

As shown in Fig. 5, We inferred that KRT23 may affect EMT progression. No matter the results for immunofluorescence showed or the images in western blot presented, the expression of E-cadherin, the epithelial relative marker was invigorated when KRT23 was knocked down while the expression of N-cadherin, vimentin, ZEB1 and β-catenin which all represented the characteristics of mesenchyme, were significantly weakened when in contrast to that in sh-NC or control group (Fig. 5A and B). Furthermore, KRT23 also regulated PI3K/AKT/GSK3β pathway. As is exhibited in Fig. 6C, when KRT23 was knocked down, the expression of PI3K, P-AKT and P-GSK3β were all decreased on protein level, while the three proteins were elevated in KRT23 group when compared with that in vector group, in spite of no significant difference for AKT and GSK3β.

KRT23 interacted with P21 to mediate HCC cell proliferation and metastasis via PI3K/AKT/ GSK3β pathway

Following we revealed that there was some interaction between KRT23 and P21 (CDKN1A) to mediate HCC progression, which was reminded by the result from STRING，a database of known and predicted protein-protein interactions(Fig. 6A). Then qRT-PCR and western blot were applied to preliminarily explore their mutual effect. As is shown in Fig. 6B (*p<0.05, **p<0.01), the expression of P21 was usually elevated when KRT23 was knocked down whether on mRNA or protein level. Subsequently, the interaction between KRT23 and P21 was further confirmed by CO-IP assay (Fig. 6C). Meanwhile, the efficiency of small interfering RNA against the sequence of P21 had been detected to be satisfied (Fig. 6D, **p<0.01).

Functionally, P21 knockdown accelerated HCC cell growth over time when compared with NC group, which can be rescued when co-transfected siP21 and siKRT23 (Fig. 7A and B, *p<0.05, **p<0.01). In colony formation assay, the number of colony formation in P21 knockdown group were more than NC group, but the increase was abolished if KRT23 knockdown was added (Fig. 7C and D, *p<0.05, **p<0.01). As for metastasis, P21 knockdown accelerated HCC cell migration and invasion and the number for migrated and invaded cells was obviously reduced in co-transfected with siP21 and siKRT23 group in contrast to that in siP21 group (Fig. 7E and F, *p<0.05, **p<0.01). similarly, the mobility of HCC cells was strengthened when P21 was knocked down. However, KRT23 knockdown distinctly impeded the activation of P21 knockdown on cell moving (Fig. 7G and H, *p<0.05, **p<0.01). In view of mechanism, the expression of PI3K, P-AKT and P-GSK3β were all improved in P21 knockdown group, which was reversed by KRT23 knockdown (Fig. 7I).

HCC with dismal prognosis has brought great burdens for public health worldwide although the various prevalence of underlying risk factors in different areas[19, 20]. Once discovered, a great deal of HCC patients have missed the best opportunity for surgical resection, following that 70% patients undertake recurrence within 5 years[19]. Furthermore, HCC is an aggressive solid tumor easily non-sensitive to chemotherapeutics applied at present[21]. Above all, it’s imperative to improve the efficacy for identification of novel biomarkers at preliminary stage of HCC and then make therapies for HCC much more cost-effective. Located on chromosome 17q21.2 and with a molecular weight of 48 kDa, KRT23 expresses a 1.65 kb mRNA and has been reported to have some function in several cancers. Xia Li revealed that KRT23 was upregulated in cervical cancer and related with immune cell infiltration, which indicted its important role in immunotherapeutic of cervical cancer[22]. It was also reported that KRT23 was highly upregulated in primary rectal cancer, which was analyzed by immunochemistry[23]. Meanwhile, KRT23 activated hTERT expression and then accelerated colorectal cancer growth[24]. In prostate basal cell carcinoma, KRT23 was recognized to be overexpressed[25]. Jian Zhou displayed that in triple-negative breast cancer high expression of KRT23 which indicated poor prognosis promoted cell proliferation[26]. As for liver disease, the expression of KRT23 in serum samples was significantly upregulated in HCC, chronic hepatitis compared to normal samples[27]. Moreover, KRT23 was investigated to be a host factor induced by HCV in liver[28]. Kim D et al. detected that KRT23 was one of proliferator–activated receptor alpha (PPARA) targeted, MYC-amplified oncogene in HCC. Taken together, all research above highlighted that KRT23 had the potential to be a diagnostic biomarker for liver cancer[29]. Our research is the first to systematically investigate the effect of KRT23 in HCC development and discover that KRT23 was overexpressed in HCC tissues and cell lines. Functionally, KRT23 knockdown suppressed HCC cell proliferation and metastasis in vitro and vivo. Above all, KRT23 might act as an oncogene in HCC.

P21, also known as CDKN1A which resided in 6p21.2[30, 31], has been investigated involvement in cell cycle regulation, differentiation, cell migration, cytoskeletal dynamic, apoptosis, transcription, DNA repair, induction of totipotent stem cells, autophagy and senescence[32, 33]. Commonly accepted is that P21 as a member of cyclin-dependent kinase inhibitors (CDKI) interacts with various cell cycle proteins to mediate G1/S growth phase arrest in response to different stimuli[34, 35]. P21 was regulated in P53 dependent or independent way at transcriptional level and also controlled by phosphorylation, ubiquitination and localization at post-transcriptional level[36]. Likewise, the function of P21 in cancer progression is rather sophisticated considering cellular environment and its subcellular location[37, 38]. In our study, analysis of STRING indicated that KRT23 might interact with P21 and KRT23 knockdown elevated the expression of P21 whether on mRNA level or protein. Co-immunoprecipitation was conducted to verify the interaction between KRT23 and P21. Furthermore, the promotion for cell proliferation, migration and invasion caused by P21 knockdown was partly reversed by KRT23 knockdown.

Epithelial- mesenchymal transition (EMT) mediated by transcriptional factors is a well-known accelerating process for neoplasia metastasis, along with mesenchymal-like morphology for easier separation from primary sites to distant organs and regulation of related biomarkers, E-cadherin for epithelial reduced while N-cadherin, vimentin and others for mesenchymal elevated[39, 40]. Rather multiple researches have elucidated that there are various signaling cascades participated in EMT, including PI3K/AKT/GSK3β pathway[41], which is reported to activated in preliminary stage of tumorigenesis and take great effect on cell aerobic glycolysis, tumor cell growth, metastasis, angiogenesis, autophagy and chemoresistance[42–44]. As the downstream of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT) is the crucial molecule in response to different stimuli and often activated by phosphorylation at different sites, most commonly at Ser 473[45]. Then activated AKT inhibits the activity of glycogen synthase kinase 3 Beta (GSK3β) via its phosphorylation at Ser 9, which generating abundant cellular glycogen for cell survival and subsequent acceleration of important gene transcription in cancer development[46, 47]. Our research unraveled that KRT23 knockdown controlled EMT process and restrained the activation of PI3K/AKT/GSK3β pathway. Moreover, activation of PI3K/AKT/GSK3β pathway caused by P21 knockdown was abrogated by KRT23 knockdown. In all, KRT23 mediated PI3K/AKT/GSK3β pathway via regulating P21. Of course, there exist some limitations for this research. Molecular mechanism in detail regarding interaction between KRT23 and P21, as well as regulation on PI3K/AKT/GSK3β pathway by KRT23 is still vague, which provides a new orientation for our following research.

In conclusion, this study revealed that KRT23 was overexpressed in HCC tissues and cell lines and mediated HCC cell growth and metastasis in vitro and vivo. Furthermore, KRT23 participated in EMT and interacted with P21 to regulate PI3K/AKT/GSK3β pathway. For convenience, the molecular mechanism for this manuscript was displayed in Fig. 8. Our research gave the orientation for KRT23 as a novel biomarker for HCC detection and even potential target molecule for clinical treatment. However, much more explorations are still needed for successful clinical application in the future.

Of course, there are some limitations in this research. Firstly, there are some differences between normal samples and tumor adjacent samples considering change for microenvironment during tumorigenesis, but it was the better choice for research ethically. Next, our study has proved the interaction of KRT23 and P21, but concrete mechanism still needs further exploration.

HCC

Hepatocellular carcinoma

KRTs

keratins

MSS

microsatellite-stable colon cancer

MSI

microsatellite-instable colon cancer

GEPIA

Gene Expression Profiling Interactive Analysis

siRNAs

Specific interfering RNAs

qRT-PCR

quantitative real-time PCR

optical density

IHC

Immunohistochemistry

CO-IP

CO-immunoprecipitation

PPARA

proliferator–activated receptor alpha

CDKI

cyclin-dependent kinase inhibitors

EMT

Epithelial- mesenchymal transition

PI3K

phosphatidylinositol 3-kinase

AKT

protein kinase B

GSK3β

glycogen synthase kinase 3 Beta

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 81502095), the Social Development, Scientific and Technological Research Project of Shaanxi Province (No. 2015SF055) and Natural Science Basic Research Program of Shaanxi(No. 2020JQ-387 ).

Authors' contributions

D G and Y L conceived and designed the experiments, W M assisted in collection of information for clinical patients and G L contacted pathological department for IHC. X Z and Mohiuddin help perform the experiments and analyze the data. M R and X L secured research funding supports. S H and P L help revised this manuscript prepared and written by D G.

Ethics approval and consent to participate

The human and animal studies involved were approved by the ethics committee of the First Affiliated Hospital of Xi'an Jiaotong University. The animal experiments were performed according to the National Institutes of Health animal use guidelines on the use of experimental animals, and is reported in accordance with ARRIVE guidelines.

Conflicts of interest

All authors have read this manuscript and agreed for publication. The authors declare that they have no conflict of interest.

Availability of data and materials

The whole data and materials supporting the conclusions were included in this manuscript.

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Table 1 is available in the Supplementary Files section.

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KRT23 acts as an oncogene in hepatocellular carcinoma by regulating PI3K/AKT/GSK3β pathway via P21

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Abstract

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Introduction

Materials and methods

Bioinformatic analysis

Cell culture and tumor tissues collection

Plasmid construction and Cell transfection assay

RNA isolation and quantitative real-time PCR (qRT-PCR)

MTT assay

Colony formation assay

Flow cytometry

Transwell assay

Wound healing experiment

The oncogenicity in nude mice

H&E staining and Immunohistochemistry (IHC)

CO-immunoprecipitation (CO-IP) and western blot

Immunofluorescence

Statistical analysis

Results

Discussion

Abbreviations

Declarations

References

Table 1

Additional Declarations

Supplementary Files

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