To the best of our knowledge, this is the first meta-analysis study that explored the expression of biomarkers under diverse pro-EMT and anti-EMT interventions in HepG2 cells. It based on the pre-intervention and post-intervention self-control of HepG2 cells. Our study incorporated 58 studies with all different ways of interventions and contained more detailed information about experimental and detection process, and excluded the poor-quality articles that did not provide details about cell origin or experimental process. Primarily, we classified these 58 studies according to the interventions, and then subgrouped some intervention groups according to the inconsisitent outcomes (enhancement or inhibition of EMT), thus systematically evaluated the effectiveness of biomarkers under interventions with different outcomes.
Our study showed that E-cadherin responds well to the intervention of medication, genetic intervention, gene knockout/knockdown, hypoxia, and other tumor microenvironments, as well as ncRNA overexpression and silencing. N-cadherin can effectively evaluate the intervention effect of medication, genetic intervention, hypoxia and other tumor microenvironments, as well as ncRNA overexpression. Vimentin reflects the effects of medication, pro-EMT genetic intervention and gene knockout/knockdown, anti-EMT ncRNA overexpression and anti-EMT ncRNA silencing and hypoxia. Snail only responds to the intervention of anti-EMT genetic intervention and gene knockout/knockdown, tumor microenvironments other than hypoxia, anti-EMT ncRNA overexpression and ncRNA silencing.
E-cadherin can better evaluate the effect of medicine, genes, microenvironment, and ncRNAs on EMT intervention. The reason may be that medicine, genes, microenvironment, and ncRNAs can directly or indirectly act on the signaling pathways to activate the upstream protein E-cadherin and therefore its expression. E-cadherin, the first member of the cadherin family, is an active inhibitor responsible for invasion and growth in many epithelial cancers [10]. Some studies have shown that E-cadherin, as a core protein, can change the adhesion activity on the cell by regulating signaling pathways including Wnt, PI3K, MAPK and Hippo in the action of tumor EMT to surface [81]. Decreased expression of E-cadherin weakens cell-cell adhesion which promotes the separation of tumor cells from the primary tumor mass, leading to invasion, proliferation, and metastasis of cancer cells [82]. It is well known that targeted drugs can directly act on tumor-associated signaling pathways. For example, Han et al. [34] reported in the meta-analysis that doxycycline significantly increased the activity of the PI3K/Akt signaling pathway and significantly inhibited the expression of E-cadherin. Another studies [83] found that after the activation of the PI3K/Akt signaling pathway, the expression of Snail, Slug, Twist, and other nuclear transcription factors was increased and that of E-cadherin was directly inhibited, thus promoting EMT. Similarly, regulation of other related pathways can also significantly decreases E-cadherin expression then induce EMT. Onder TT et al. [84] found that the expression of inducible transcription factors Zeb, Snail, and Twist could lead to the downregulation of E-cadherin. Gregory PA et al. [85] found that the miR-200 family and miR-205 could inhibit transcription factors ZEB1 and SIP1 and then cause the decreased expression of E-cadherin, thus inducing EMT. In addition, hypoxia can promote the EMT of tumor cells via HIF-1α [86]. HIF-1α is the upstream regulator of Snail [87]. It can directly induce the increase of snail expression. In conclusion, E-cadherin can effectively reflect the EMT process in HepG2 cells under various interventions. Therefore, we recommend it to be the first choice in future in vitro studies as a EMT biomarker for medicine, gene, microenvironment, and ncRNA interventions in HepG2 cells.
Our study also showed that N-cadherin expression changes significantly to EMT process induced by medicine, genetic intervention, tumor microenvironment and ncRNA overexpression intervention. N-cadherin is a calcium-dependent single-chain transmembrane glycoprotein that mediates homotype and heterotype adhesion between cells [88]. Overexpression of N-cadherin changes cell polarity and inter-cell adhesion, making cancer cells more prone to metastasis [89]. A previous study showed that during EMT, cadherin changes from E-cadherin to N-cadherin, so the upregulated expression of N-cadherin is considered an important EMT biomarker [90]. N - cadherin expression in tumor cells was regulated by TGF - β1, Wnt/β- catenin, EGFR and NF-kappa B, etc [88]. Jiao, M et al. [63] found that the PI3K/Akt/HIF-1 pathway plays a key role in the EMT of HepG2 cells under hypoxic conditions, inhibiting the expression of E-cadherin and promoting the expression of N-cadherin and vimentin. However, the changes of N-cadherin expression in gene knockout/knockdown, ncRNA silence group were not statistically significant, indicating that N-cadherin may be regulated by multiple genes rather than single factor. Medicine and microenvironmental intervention could acts on multiple regulatory factors, and then affect the expression of N-cadherin. No single gene has provened to cause significant changes on N-cadherin. It could be also related to the following aspects: (1) the amount of research into the three kinds of the group were less (2–3); (2) part of the included trials [55, 75–76] did not report the dose and duration of the intervention, which may also be one of the reasons for greater heterogeneity and inconsistence among the studies; (3) it may be that the types of genes and ncRNAs involved in the included experiments were not consistent, as well as the complex signaling pathways such as TGF-β1, Wnt/β-catenin, EGFR, and NF-κB ultimately led to significant differences in the efficacy of the intervention and resulted in greater heterogeneity among the included studies. Therefore, we suggest the following: (1) when researchers select medicine, genetic intervention, and tumor microenvironment, and overexpression of ncRNA to influence the EMT effect on HepG2 cells, N-cadherin can be used as a secondary marker; (2) More studies involving the expression of N-cadherin regulated by gene knockout/knockdown and ncRNA silence are needed to establish the research system of gene/ncRNA-N-cadherin-signaling pathway to reduce the heterogeneity and improve the conversion and utilization.
We also found that medicine, pro-EMT genetic invervention, gene knockout/knockout, anti-EMT ncRNA overexpression/silencing, and hypoxia significantly affects Vimentin. Expression of Vimentin increases as epithelial cells transforms to mesenchymal cells during the EMT process [91]. Liu et al. [94] showed that Vimentin participates in the EMT of cancer cells by mediating cytoskeletal tissue and local adhesion maturation. Vimentin is upregulated in HCC and then induced in the EMT process [92]. Dan et al. [93] found that Vimentin acetylation is involved in SIRT5-mediated migration in liver cancer. Most antitumor drugs can inhibit the growth and invasion of tumor cells by directly or indirectly inhibiting Vimentin. Satelli A et al [94] found that a Vimentin-binding mini-peptide can bind to Vimentin, target, and interact with Vimentin to interfere with various signaling pathways such as Erk, AKT1, Axl and PI3K and then cell functions. Liu et al. [27] also reported that Fucoidan inhibited the expression of vimentin by inhibiting the activity of the PI3K/AKT signaling pathway. However, interventions for anti-EMT genetic intervention, tumor microenvironments other than hypoxia, pro-EMT ncRNA overexpression/silencing does not significantly change Vimentin expression. In conclusion, Because of the limitation in the number of included studies (1–2), there is not enough data to support the evaluation effect of Vimentin on the above three interventions. In summary, we believe that the following: (1) Vimentin could be a valuable marker for interventions of medicine, pro-EMT gene overexpression/knockdown/knockout, hypoxia, anti-EMT ncRNA overexpression/silencing act on HepG2 cells to affect the EMT; (2) Future studies should pay more attention to the effects of intervention for tumor microenvironments other than hypoxia, pro-EMT ncRNA overexpression/silencing needs further investigation.
In terms of Snail, interventions of tumor microenvironments other than hypoxia, anti-EMT gene overexpression, gene knockdown/knockdown, anti-EMT ncRNA overexpression, and ncRNA silencing induced significant changes of Snail expression. Snail family of proteins is one of the most important EMT downstream signaling pathway transcription factors. It’s a key inducer of EMT, which can regulate invasion and migration [95]. The possible reasons are as follows: During EMT, its downstream signaling pathway transcription factors (EMT-TFS) are activated, mainly including zinc finger transcription factors family (Snail1 and Snail2), Twist family (Twist1 and Twist2), and ZEB family related to zinc finger E-box (ZEB1 and ZEB2), etc. Among them, the Snail family of proteins is a key inducer of EMT, which can regulate invasion and migration [95]. Its main role is to directly binds to the E-Box sequence located in the promoter region of the E-cadherin gene and inhibit its transcription [96], hence promoting EMT. There are many studies about the relationship between the Snail pathway and EMT [97–98], but few studies evaluated its potential as EMT biomarker. (Only 21 articles were included in this paper) Therefore, we believe that Snail could be used as a reference marker in future studies to further investigation are needed to explore its value under various interventions.
Our strength is that we included all different kinds of interventions and performed subgroup analysis to reduce the effects of heterogeneity between studies. The limitation is the heterogeneity of in vitro study, intervention dose and processing time, cell source, culture medium, etc. 65% of the included studies did not provide the concentration of the intervention. That’s why SMD was used in the pooled analysis of the data. In addition, the different sources, culture details and intervention time of HepG2 cells in the included experiments all had a certain effect on the subsequent analysis. Therefore, it is suggested that the origin, culture, and intervention time of HepG2 cells should be clarified and unified in relevant studies tocould help reduce the heterogeneity among studies. Moreover, more attention should be paid to standardization and adequate reporting of the dose and unit of interventions and determining cell viability in in vitro experimental studies so as to further improve the transparency of cell experimental research. This would eventually improve the utilization value of basic research and provide a reliable theoretical basis for the transformation from basic research to clinical research.
In summary, biomarkers work differently under various intervention in HepG2 EMT model. E-cadherin, N-cadherin, and Vimentin respond well to medicine intervention. E-cadherin work well under genetic intervention. E-cadherin and N-cadherin reflects tumor microenvironment intervention. Under ncRNA intervention, the expression of E-cadherin, change significantly. Moreover, it is necessary to further standardize the implementation of cell culture and intervention measures in vitro, especially the standardized procedure for the time and dose of various interventions to improve the transparency of the whole process of in vitro cell research and promote its transformation and utilization value.