In addition, we generated dermo–epidermal substitutes using freshly isolated and sorted basal iβ4+ or suprabasal iβ4− keratinocytes and transplanted them on immuno-compromised rats. We show that suprabasal iβ4− keratinocytes acquire a similar proliferative capacity as basal iβ4+ keratinocytes after two weeks of culture in vitro, with expression of high levels of iβ4 and downregulation of K10 expression. In addition, both basal iβ4+ and suprabasal iβ4− keratinocytes acquire authentic self-renewing properties during the in vitro 3D-culture phase and are able to generate and maintain a fully stratified epidermis for 16 weeks in vivo. Therefore, against the leading dogma, we propose that human suprabasal keratinocytes can retro-differentiate into true basal stem cells in a wound situation and/or when in contact with the basement membrane.The basal layer of human interfollicular epidermis has been described to harbour both quiescent keratinocyte stem cells and a transit amplifying cell population that maintains the suprabasal epidermal layers. We performed immunofluorescence analyses and revealed that the main proliferative keratinocyte pool in vivo resides suprabasally. We isolated from the human epidermis two distinct cell populations, the basal and the suprabasal keratinocytes, according to the expression of integrin β4 (iβ4). We compared basal iβ4 Keratinocytes represent 90–95% of the total cell population in the human interfollicular epidermis (IFE) . They arNevertheless, identity and localization of self-renewing keratinocytes in the IFE are still largely debated . Early rIn summary, these reports accredited two main models : basal uOur investigation systematically characterizes the biological and biochemical properties of two distinct, however, adjacent, populations of keratinocytes in human skin. Additionally to common immuno-histochemical methods, we implemented Raman spectroscopy  as a nonHistological haematoxylin/eosin and immunofluorescence staining on tissue sections are described in detail by Biedermann et al. . All theHuman skin samples were obtained from the Department of Surgery of the University Children’s Hospital in Zurich. Parents or patients gave informed consent. This study was conducted according to the Declaration of Helsinki Principles and after permission by the Ethics Commission of the Canton Zurich (BASEC Request No. 2018-00269).−1 dispase  dissolved in Hank’s balanced salt solution containing 5 mg mL−1 gentamycin  at 4 °C. The epidermis and dermis were subsequently separated using forceps. The epidermis was digested in 1% trypsin and 5 mM EDTA for 3 min at 37 °C (Thermo Fisher Scientific). Epidermal cells were passed through a 100 µm cell strainer , washed in phosphate buffered saline  and resuspended in serum free keratinocyte medium CT57  containing bovine pituitary extract, epidermal growth factor and the addition of 5 µg mL−1 gentamycin. For keratinocyte selection, the epidermal cells were cultured on bovine collagen I  coated culture flasks. For gaining fibroblasts, the dermal tissue was further digested in 2 mg mL−1 collagenase blend F  for 1 h at 37 °C. After mechanical disruption by pipetting, lysis of red blood cells in RBC lysis buffer according to the instruction of the manufacturer (Sigma) and washing in PBS, the dermal fibroblasts were plated on uncoated culture flasks and grown in DMEM containing 10% foetal calf serum (FCS), 4 mM l-alanyl-l-glutamine, 1 mM sodium pyruvate and 5 mg mL−1 gentamycin . The medium was changed every 2–3 days and cells were passaged when approximately 80–90% confluence was reached.Human epidermal keratinocytes and dermal fibroblasts were isolated and cultured according to Pontiggia et al. . Briefly3 freshly isolated keratinocytes were concentrated onto a microscope slide by cytospin centrifugation . After fixation for 3 min in aceton:methanol 1:1, the cells were stained with the Ki67 and K10 antibodies as mentioned above. For each sample (n = 5), we analyzed three different microscopic frames which were taken randomly. We calculated which percentage of the total number of Ki67 positive cells was Ki67-single positive  or Ki67/K10-double positive . Means, standard deviations, and significances (unpaired t test) were calculated using GraphPad Prism . Significance was set at an alpha-level of 0.005. In few cases, we stained additionally for K15 and iβ4 to double-check the identity of basal cells and for illustrative purposes.For quantification of the number of basal- and suprabasal-cycling (Ki67 positive) keratinocytes, 30 × 106 cells per ml in cell culture medium CT57 containing the iβ4-Alexa Fluor 488 antibody (see above). In some experiments, iα6-PE or Zenon pre-labelled K10 antibodies were used (see above). In addition, the Zombie Aqua Viability Dye  was added according to the instruction of the manufacturer. The cells were incubated on ice for 20 min. Before centrifugation, the cell suspension was adjusted to 4 mL with PBS and underlaid with 1 mL fetal calf serum (Invitrogen). Finally, the cells were resuspended in CT57 and transferred into a centrifugation tube through a 50 µm cell strainer cap . Flow cytometric analysis and sorting was performed at the Center for Microscopy and Image Analysis of the University of Zurich on a FACS Aria III  equipped with 405 nm (violet), 488 nm (blue), 561 nm (yellow/green) and 635 nm (red) lasers and using the 85 µm nozzle. Sorted cells were collected in CT57 medium with 50% FCS, washed once in PBS, counted and either plated on collagen I-coated dishes and further cultured in CT57 medium, or directly seeded on dermal substitutes  were washed twice in PBS and stored at −80 °C until further processing. Cells from 4 different donors were collected. Peptide samples were prepared according to established protocols [g, 20 min, RT) and the filtrate was collected. The filtrate was then acidified with 5% trifluoroacetic acid (TFA) to a final concentration of 0.5%. After clean-up, samples were resuspended in 3% acetonitrile and 0.1% formic acid then cleaned up using C18 stage tips (Pierce), following manufacturer’s instructions. Samples were analyzed on a Q-Exactive HF Orbitrap LC-MS/MS mass spectrometer (Thermo Fisher Scientific). The device was run in data-independent acquisition (DIA) mode using XCalibur control software (Thermo Fisher Scientific). Automated quality control was conducted every 3 samples. Data and sample annotation was done using b-fabric (Available online: https://fgcz-bfabric.uzh.ch/bfabric/ (accessed on 15 June 2020). Identification was carried out using MASCOT (Matrix Science), using decoys. Protein quantification of the DIA data sets was carried out using Spectronaut . Filters for the generation of volcano plots are set to ≥1.5 absolute fold change. All p-values are corrected for multiple testing [100,000 freshly isolated and sorted keratinocytes  (accessed on 10 June 2022) via the Proteomics Identifications Database (PRIDE) partner repository with the data set identifier PXD034543.Raw mass spectrometry proteome data and search results have been deposited to the ProteomeXchange Consortium (+ and iβ4−), were seeded on mitomycin (Sigma) treated 3T3-Swiss albino mouse feeder fibroblasts (ATCC Number: CCL-92) in six-well culture plates. The experiment was run in triplicate for each cell type for 8 different donors. After 10 days, single colonies were visible. The colonies were washed in PBS, fixed in aceton:methanol (1:1) for 5 min and stained in 0.2% trypan-blue solution (Sigma) for 5 min. After thorough washing, pictures of the plates were taken by standard light microscopy in a G:BOX Imaging System , the colonies were manually counted with the Cell Counter plugin of Fiji [t test) were calculated using GraphPad Prism. Significance was set at an alpha-level of 0.05.The colony-forming assay was performed according to Pontiggia et al. , with so of Fiji  and stat®, microPhotonX GmbH, Tutzing, Germany) with an inverted microscopic setup. A diode laser of 785 nm wavelength and 80 mw power  is used for Raman excitation. All Raman scattered photons are collected by a 60-fold water immersion objective   with correction collar set to 0.17 mm. The detection of the Raman scattered photons is achieved using a diffraction grating and a Charge-Coupled Device detector . 25 μL of PFA-fixed cells in PBS buffer were pipetted into a sterile μ-channel Slide with a 0.17 mm thick borosilicate glass bottom. 60 spectra were collected from the cytoplasmic domain of the cells. Cells were exposed to the laser for 15 s to collect a single Raman spectrum. Cells were trapped by the laser focal point during the measurements using optical trapping capability. Statistical analysis of Raman data: Processing of the spectral data and statistical analysis are performed using CT-RamSES software . All Raman spectra were cropped to the spectral range of 350–1800 cm−1, which contains most of the biological-relevant spectral bands. This was followed by a baseline correction using an asymmetric least square fit, cosmic spikes were removed, and the spectra were smoothened with a median filter. Next, the spectra were interpolated to continuous wave numbers and normalized by implementing a unit vector normalization. Principal Component Analysis (PCA) was then applied on the datasets. PCA is a multivariate statistical analysis method used to reduce the dimensions of large data sets by transforming the large data variables into smaller numbers of components called Principal components (PCs). The first principal component (PC1) explains for the largest possible variance in the data set (most important variances), the PC2 explains the second most important, etc. PCA was performed under Python 3, using the scikit-learn package. PCA Score plots can illuminate the differences and similarities among samples, while the PCA Loadings display the Raman spectral differences that are used to compare the analysed samples. Hierarchical cluster analysis (HCA) was applied as an unsupervised statistical analysis and clustering method, to separate the Raman spectra based on the different spectral observations to different clusters. Similar to PCA, linear discriminant analysis (LDA) was applied to characterize differences between samples. However, LDA is a stronger tool for samples discrimination than PCA. In LDA, linear transformations of the variables to the new discriminant functions (LDs) are computed, in which the directions of the LDs are determined in the spectral space [Raman measurements are implemented using Raman microscopic laser trapping system  and 0.4 mL DMEM containing 50 × 103 fibroblasts. After polymerization (5 min at room temperature and two hours at 37 °C), the dermal substitutes were compressed to 1 mm thickness according to Braziulis et al. [6 freshly isolated and sorted keratinocytes in 1.5 mL CT57 were seeded onto each dermal equivalent. Triplicate wells were set up for each dermo–epidermal substitute. The constructs were cultured with DMEM in the lower chamber and CT57 in the upper chamber with daily medium change. After 7 days, the dermo–epidermal substitutes were ready for transplantation.Organotypic cultures were prepared using a previously established transwell system consisting of 6-well cell culture inserts with membranes of 3.0 µm pore-size  . The col−1 body weight, TemgesicTM, Indivior AG, Baar, Switzerland) was applied for analgesia. Dressing changes and photographic pictures were made once a week. Rats were monitored for the entire length of the experiment. At week 16 post transplantation, animals were sacrificed, and transplants were excised and embedded either in paraffin or TissueTek  for histological and immunofluorescence analysis. A total of 6 rats received a dermo–epidermal skin substitute formed with iβ4+ keratinocytes and 10 rats with iβ4− keratinocytes.Animal studies were approved by the local committee for experimental animal research (license number ZH90/15 and ZH45/19) and performed as previously published . Ten weestratum basale  were identified among non-proliferative keratinocytes  and integrin α6 low-expression (iα6low) keratinocytes in the epidermis by FACS analyses  as the most popular for further investigations. We first confirmed the presence of integrin α6 high-expression  and melanoma antigen (expressed in melanocytes)  .+ and iβ4− cells in vitro directly after isolation or after two weeks culture time. + and iβ4− and then immediately centrifuged to a cytospin slide. As expected, at time point 0 h, exclusively basal (iβ4+) cells expressed iβ4 (green in −) cells expressed K10  skin in pulation E,F, red.division G,H, 24 hrly well G,H, 5 d.ress iβ4 I,J. The  culture K,L, red.We analysed the biochemical–molecular properties of basal and suprabasal keratinocytes by Raman spectroscopy.+ and iβ4− keratinocytes, just after sorting (day 0) or after 7 days of culture (day 7). Raman mean spectra (n = 60) at day 0 showed distinctive variations in the biochemical composition between the two cell types (−1 (C–H twisting and scissoring vibrations) in the case of suprabasal iβ4− keratinocytes compared to iβ4+ basal keratinocytes  then 10 days after seeding, the number of formed colonies was counted (+ cells formed significantly more colonies (41.9% ± 10.2) than suprabasal iβ4− cells (5.7% ± 4.8). Yet, after pre-culturing for 15 days, significantly more iβ4− cells were able to produce a colony (24.2% ± 12.7), so that the difference to iβ4+ cells was not significant anymore (p > 0.05). Unsorted cells, as a mixture of both cell types, produced always intermediate results (17.7% ± 5.7 and 33.2% ± 11.7).We also compared the colony-forming efficiency of unsorted keratinocytes and sorted iβ4 counted A–C. Alte counted D,F. Figu+ and iβ4− keratinocytes displayed high proliferative and colony-forming properties, we investigated whether they would develop into a multi-layered stratified epidermis in vivo. Hence, we used freshly isolated and sorted iβ4+ or iβ4− cells (which were never in contact with culture plastic) to prepare dermo–epidermal skin equivalents which were transplanted and maintained on the backs of immune-incompetent rats for up to 16 weeks. We observed that both iβ4+ and iβ4− keratinocytes had equal capacity to develop into a stratified epidermis. Since both iβ4− constructs (Immunofluorescence staining for keratin 10 (K10) revealed the presence of a K10-negative basal layer in both iβ4+ and iβ4nstructs C–F and instructs , as wellnstructs G,H and Knstructs , revealenstructs , desmoglnstructs . Ki67 stnstructs E,F.+ or iβ4− keratinocytes did not express K16 anymore after 16 weeks of transplantation . We sorted two keratinocyte populations according to the expression of the iβ4 chain. iβ4 forms heterodimers with iα6 iα6, CD49f) which in turn is a biomarker commonly found in more than 30 different populations of SCs . The iα6f which i+ keratinocytes are fast cycling, and iβ4− cells are post-mitotic differentiating keratinocytes with limited proliferative ability [+ keratinocytes are induced to vigorously proliferate after their dissociation into single cells and culture in vitro. In contrast, we show here that suprabasal iβ4− keratinocytes (manifestly more proliferative in homeostatic human epidermis but devoid of basement membrane adhesion molecules) needed two weeks to acquire a similar proliferative capacity as basal iβ4+ keratinocytes in vitro. In this period of time, terminally differentiated cells (possibly granular and upper spinous cells) may not adhere to the cell culture dish, introducing a selective enrichment of those early differentiated suprabasal keratinocytes which maintain a potential to de-differentiate back to basal stem cells. This may happen both during the 2D-culture on plastic dishes before performing proliferation, colony-forming and Raman spectral analyses and during the 3D-organotypic culture in vitro before transplantation. Due to the high purity of the sorting procedure shown in + keratinocytes overgrew the culture of sorted iβ4− keratinocytes in the short time before in vitro analyses or before transplantation. Also, we did not observe any particular cell dying in the sorted, plated, and adherent iβ4− keratinocytes in the 2D-cultures. We assume instead that iβ4− keratinocytes pass through a re-programming phase in the culture (both in 3D-skin substitutes and 2D-culture on plastic), which includes the activation of the genes for integrin receptors and, conceivably, other (adhesion) molecules and the deactivation of genes for differentiation proteins. Yet, once the proliferation is triggered, both isolated and cultured basal and suprabasal keratinocytes (1) express high levels of iβ4 and downregulate K10 expression, (2) demonstrate similar colony-forming efficiencies, (3) express analogous phenotype by displaying similar Raman spectral fingerprints, (4) establish a basement membrane and a functional niche, and, finally and most important, (5) both iβ4+ and iβ4− keratinocytes develop into a epidermis in skin substitutes, which is maintained for at least 16 weeks in a pre-clinical animal model. Therefore, based on these results, we believe to observe that in normal interfollicular skin, the biological  and biochemical (Raman fingerprint) properties of basal and suprabasal cells after culture in vitro become similar, which may reflect a similar regenerative potential.Some previous in vitro studies on the human epidermis showed that iβ4 ability ,53. Our + iβ4− suprabasal keratinocytes can retro-differentiate into active or even quiescent SC when they face a wound situation and/or when they get into contact with the basement membrane  one in the basal layer and an active (fast cycling) one in the suprabasal compartment. Consequently, we postulate that K10membrane .bri CD71dim, in our work iβ4+), transit amplifying  and early differentiating  keratinocyte populations and evaluated them in transplantation experiments. They observed a gradually dropping regenerative capacity and conceived of a continuum rather than a black and white transition between the keratinocyte stem and differentiating cells. The authors therefore speculate on the existence of cells with varying degrees of self-renewing capacities [Interestingly, Schlüter et al.  analysedpacities  which coSimilarly, after single cell-RNA sequencing, Wang et al. described four spatially different SC populations located at the top or in the tip of rete ridges in the basal epidermal layer . They suIn fact, both studies ,55 do noIn support of this hypothesis, some other authors found indications of a possible de-differentiation capacity of committed suprabasal keratinocytes which would allow them to re-acquire “stemnes” properties: Fu et al. treated patients that had leg ulcers with topical recombinant human epidermal growth factor and observed the induction of expression of the stem cell marker K19 in the spinous and granular layer of the regenerated epidermis . SimilarTrying to find a synthesis of many observations made in different tissues, such as the gut, hair follicle, bone marrow, and brain, Li et al. proposed a general model which implies the existence of two stem cell populations, referred to as quiescent and active stem cells, being located in separate yet adjoining locations, the basal and the suprabasal compartment, respectively ,61. TheyWe propose that this could also be true for the human epidermis , even thIn conclusion, our data show that basal and suprabasal keratinocytes own self-renewing potential and, after 3D-organotypic or 2D cultivation in vitro, can generate and maintain a stratified epidermis for many weeks in vivo. We suggest that the interplay between basal quiescent and suprabasal active keratinocytes stem cells is part of a self-renewal system for the maintenance and damage repair of the adult human interfollicular epidermis during homeostasis.


Classic fail-safe N and Egger's test to test for publication bias and for moderation with the homogeneity tests. The results showed a negative association between quality of parent-child relationship and problematic internet use . The moderation analysis found that compared with internet addiction tendency, the association between social media addiction and parent-child relationship was stronger. Moreover, the association between the parent-child relationship and problematic internet use of emerging adults (18–25 years old) was stronger than that of adolescents (12−18 years old). Furthermore, the negative association between parent-child relationship and problematic internet use was weaker (a) in Italy than those in Turkey and China, (b) when using CPS , IPPA (Inventory of Parent and Peer Attachment), or PARQ (Parent-Child Relationship Questionnaire) measuring parent-child relationship than using PCCS , (c) when using IAT measuring problematic internet use rather than using IGDS or APIUS. Hence, these results indicate a negative association between parent-child relationships and problematic internet use, and the association is moderated by types of problematic internet use, age, country, scales of both parent-child relationship and problematic internet use.As past studies of the association between parent-child relationship and problematic internet use show mixed results and are influenced by many factors, this meta-analysis of 75 primary Chinese and English language studies from 1990 to 2021 with 110,601 participants (aged 6−25 years) explored (a) the overall association between parent-child relationship and problematic internet use, and (b) whether the association is affected by their types, country, measures, objects of the parent-child relationship, gender, age, year and publication types. We used funnel plots,  With the rapid development of information technology and lifestyle changes, the number of Internet users is increasing  and literature types , see The definition and terminology of problematic Internet use are inconsistent in different research   is higher than that in other countries, and shows an upward trend   had a greater impact on problematic Internet use than social factors . Among social factors, parent-child relationship and peer relationship had a more significant impact on problematic Internet use ”, “parent-child affinity (亲子亲和)”, “parent-child attachment (亲子依恋)”, “parent-child communication (亲子沟通)”, “parent-child relationship (亲子关系)”, “mobile phone addiction (手机成瘾)”, “internet game addiction (网络游戏成瘾)”, “internet addiction (网络成瘾)”, “mobile phone dependence (手机依赖)”, “pathological internet use (病理性网络使用)”, “excessive internet use (网络过度使用)”, “compulsive internet use (强迫性网络使用)”, “problematic social media use (问题性社交媒体使用)”, “social media addiction (社交媒体成瘾)” and “problematic mobile phone use (手机使用问题)” were searched in the following Chinese databases. The China National Knowledge Infrastructure (CNKI) retrieved 215 papers, and the China Science and Technology Journal Database retrieved 91 studies. Wanfang Data Knowledge Service Platform retrieved 144 studies, a total of 450 studies.Second, the topics retrieved in the following seven English databases were “parent-child conflict”, “parent-child affinity”, “parent-child attachment”, “parent-child communication”, “parent-adolescent communication”, “parent-child relationship”, “parental Attachment”, “parent-adolescent relationship”, “parents” phubbing”, “mobile phone addiction”, “internet game addiction”, “internet addiction”, “mobile phone dependence”, “pathological internet use”, “excessive internet use”, “compulsive internet use”, “Facebook addiction”, “social media addiction”, “problematic social media use” and “problematic mobile phone use”. Web of Science (SCI) retrieved 298 literature articles, Proquest retrieved 44 articles, Science direct retrieved 94 articles, PsycNet (APA) retrieved 29 articles, SAGE retrieved 3567 articles, JSTOR retrieved 599 articles, and Scopus retrieved 49 articles, a total of 4680 literature articles.This study was conducted under the guidance of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)  Only cross-sectional studies were included, longitudinal studies were not included; (b) only studies using the scales as a research tool were included; (c) the empirical quantitative studies includes Pearson's product-moment coefficients es Card, , (d) stues Card, , this evFinally, 75 studies meeting the criteria were included, including 34 Chinese studies, 41 English studies, 11 master's and doctoral theses, and 64 journal articles, with 79 independent sample groups and 110,691 participants.r-values of each dimension of the scales, the average value was taken as the effect size for the overall correlation coefficient. For the scales of reverse scoring, r-values were converted by sign. (c) The types of parent-child relationship and problematic internet use were coded according to the traits actually measured on the scales. (d) If multiple effect sizes on the strength of the association between parent-child relationship and problematic Internet use in the same sample, only the overall effect size was used. (e) Year is the actual research year to encode.The coding criteria for studies that met the inclusion criteria were as follows: author, year, types of literature, types of problematic Internet use, types of parent-child relationship, age, gender, country, parental identity, scales, and correlation coefficient between parent-child relationship and problematic Internet use see . The folThe coding standards of the regulating variables are as follows. According to the severity of problematic Internet use Davis,  and the We also tested differences in the association between the quality of parent-child relationship and problematic Internet use across countries. Because different scales have different standards, most of the measured characteristics coincide with each other. Therefore, multiple versions of the same scale are coded as one type. Only the scales used more were coded, and the scales used less were coded as other types of scales. Parental Identity in the Parent-child Relationship was coded as the father, the mother and the parents. Gender was coded according to the sex ratio of boys recorded in the studies. Age groups were coded as children (012 years old), adolescents (12−17 years old) classic fail-safe N and Egger's test  and published  studies were included. In addition, the funnel plots, p ≤ 0.05); otherwise, a fixed effect model was used. Furthermore, if the Q test was significant and I2 > 75, it indicates there are heterogeneous among effect sizes, then main effect analysis and moderating effect analysis were analyzed  = 3017.16, I2 = 95.92, p < 0.001] and I2 75, indicating a high degree of heterogeneity among the effect sizes. Hence, the random effect model was selected for the meta-analysis.The results of the heterogeneity test performed on the effect sizes in this study showed that the included effect sizes were heterogeneous , Z = −14.35, Q(123) = 3017.16, p < 0.001). According to the criteria proposed for quantitative analysis, 0.1, 0.2 and 0.3 are regarded as small-level, middle-level, and large-level correlation coefficients, respectively . See Classic fail-safe N showed Z = −67.11, p < 0.05 and Egger's test showed t = 1.09, p > 0.05. Therefore, the possibility of publication bias in this study was extremely low.The funnel plot see  shows thSubgroup analyses and meta-regression were used to test whether the strength of the association between parent-child relationships and problematic Internet use was influenced by the moderate indicators.Subgroup analysis was conducted on the moderating variables of types of literature publication, types of problematic Internet use, types of parent-child relationship, age, country, parental identity and scales.Q (4) = 7.02, p > 0.05]. Further homogeneity test found that the association between SMA (r = −0.27) and the parent-child relationship was significantly stronger than the association between EIU (r = −0.12) and the parent-child relationship  (see a) The homogeneity test found that the types of problematic Internet use did not moderate the negative link between parent-child relationship and problematic Internet use [.05] see .Q (4) = 75.67, p < 0.001]. However, the further homogeneity test indicated that the association between parent-child communication (r = −0.23) and problematic Internet use was stronger than the association between parent-child affinity (r = −0.10) and problematic Internet use  (see b) The types of parent-child relationship significantly moderate the link between parent-child relationship and problematic Internet use [.01] see .r = −0.22) was stronger than in adolescents (r = −0.17)  (see c) Age did not moderate the association between parent-child relationship and problematic Internet use. But the association between parent-child relationship and problematic Internet use in emerging adults (.05] see .Q (3) = 5.99, p > 0.05, see r = −0.13) was significantly weaker than in Turkey (r = −0.24)  or approached significantly weaker in China (r = −0.19) .d) Country did not moderate the association between parent-child relationship and problematic Internet use  (see e) Parental identity of parent-child relationship did not moderate the association between the parent-child relationship and problematic Internet use [.05] see .Q (4) = 25.65, p < 0.001, see r = −0.29) than using the CPS (r = −0.18) , the IPPA (r = −0.21) , the FACES (r = −0.20) , or the PARQ (r = −0.19) .f) The parent-child relationship scales significantly moderated the association between the parent-child relationship and problematic Internet use  or the APIUS (r = −0.22) . The association between parent-child relationship and problematic Internet use using the IGDS (r = −0.22) was significantly stronger than using the other scales (r = −0.15) .g) The problematic Internet use measures significantly moderate the association between parent-child relationship and problematic Internet use  = 1.46, p > 0.05) and gender  did not significantly moderate the association between parent-child relationship and problematic Internet use.Meta-regression analysis of year and gender found that see  year   was stronger than that of in this study  than China (20 points) and Turkey (37 points), and Italians are less connected to their families than those in China and Turkey. Countries with higher levels of individualism have higher subjective wellbeing; they will be happy if they are different from the group. Italian parents cultivate their independence, encourage their children to express themselves, and use authoritative education methods  Integrating studies that examined parent-child relationships from different aspects, it seems that compared with parent-child attachment and general types of parent-child relationships, parent-child communication was more important for the impact of problematic Internet use. This conclusion provides a direction for future intervention in the parent-child relationship and can further prevent the problematic Internet use by reducing the pressure on the surrounding socialenvironment.b) Among different types of problematic Internet use, the association between SMA/IGD and the parent-child relationship was stronger, which indicated that it is necessary to further clarify the types of problematic Internet use and identify the differences among different types of problematic Internet use. A meta-analysis also found this difference  This study did not strictly distinguish between father-child relationship and mother-child relationship between SMA and IGD. There were more females with SMA and more males with IGD  Studies on the association between emerging adults' problematic Internet use and the parent-child relationship should be strengthened. Emerging adults are in a stage of delayed identity, and when exploring life and self, the quality of social support (parent-child relationship) is very important to them. The availability of the Internet to emerging adults might also make them more prone to problematic Internet use. At the same time, it is also essential to strengthen the monitoring of problematic Internet use rates in countries with high power distance. The parent-child relationship may be more important, providing direction for futureintervention.e) Currently, there are various scales for measuring parent-child relationship and problematic Internet use  The Internet use disorder model (Ceyhan et al., In conclusion, this study is the first to systematically examine the effect sizes of the association between parent-child relationship and problematic Internet use. Parent-child relationship was negatively correlated with problematic Internet use, and it was moderated by types of problematic internet use, age, country and scales of parent-child relationship and problematic internet use.The current meta-analysis involving 114,098 subjects and 79 independent samples worldwide provides an accurate effect size of the differences from a global perspective. The association between social media addiction and parent-child relationship is stronger than the association between excessive Internet use and parent-child relationship. Moreover, the association between the parent-child relationship and problematic Internet use in emerging adults was significantly stronger than that in adolescents. Findings also suggested the association between the parent-child relationship and problematic Internet use was weaker in Italy than in Turkey or China. In addition, different measures have different effects on the association between the parent-child relationship and problematic Internet use. These results suggested that it is necessary to combine culture to define and study problematic Internet use in the future more accurately.The original contributions presented in the study are included in the article/YZ: methodology, investigation, writing-original draft, data curation, formal analysis, visualization, writing-original draft, and writing-review & editing. LD: conceptualization, methodology, formal analysis, writing-original draft, writing-review & editing, project administration, and funding acquisition. KW: writing-original draft and writing-review & editing. All authors approval of the version to be published and agreement to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The retrieval practice effect refers to the fact that one or even multiple retrievals of memory content during the same period are more effective than repeated studying to promote future memory retention. It is effective for numerous declarative knowledge learning materials. However, studies have demonstrated that retrieval practice does not benefit problem-solving skill learning. This study used worked examples from math word problem tasks as learning materials, considering the retrieval difficulty as the main factor. Experiment 1 explored the effect of retrieval practice on acquiring problem-solving skills under different initial testing difficulties. Experiment 2 manipulated the difficulty of materials as a variable to ascertain the effect of retrieval practice on problem-solving skills under different material difficulty levels. Experiment 3 introduced feedback variables to facilitate the generation of the retrieval practice effect and examined the effects of various difficulty feedback levels on problem-solving skills learning. Results showed that, compared with restudying examples (SSSS), the example-problem pairs (STST) did not promote delayed test performance. As for the retrieval practice effect, as no differences or advantages were found in the repeated study group on the immediate test, the retrieval practice group generally outperformed the repeated study group on the delayed test. However, across the three experiments, we found no evidence of retrieval practice affecting results during an enhanced delayed test. Therefore, there may be no retrieval practice effect on acquiring problem-solving skills from worked examples. The scoStudies have demonstrated that retrieval practice does not benefit problem-solving skill learning . Worked Retrieval practice after a worked example study does not enhance delayed problem-solving performance compared to restudy. Learning from worked examples involves procedural knowledge; thus, for novices, it poses a high learning challenge, has a heavy cognitive load, and is difficult to understand. Therefore, the retrieval practice effect is promoted by reducing learning difficulty. There are two types of connection methods between examples and problems. In addition to the example-problem pairs, an incomplete example  may be added . StudiesIn addition to reducing the difficulty, feedback can promote the retrieval practice effect . Giving 2.Experiment 1 investigated the effect on the immediate and delayed retention test performance of the restudy example, example-problem pairs, and incomplete examples. As compared to the restudy example condition, the retrieval practice condition  required more mental effort and time . So pote2.1.2.1.1.M = 19.58, SD = 3.85) who did not take part in similar experiments before. They were randomly assigned to one of six conditions (n = 20 per condition) resulting from a 3 × 2 factorial design with between-subjects factors initial test conditions  and final test time . The dependent variables were the final test scores and mental effort scores.The participants were 120 Minnan Normal University students  concepts were used as examples in the study. The learning conditions were divided into three groups: the restudying examples, the example-problem pairs, and the incomplete example. The learning material was four similar examples. “Similar materials” refer to the same structure but different surface features. It means that the problems described in the text are different, but the calculation steps are same  The daily growth rate of grass is: (10 * 20 − 15 * 10) / (20 − 10) = 5. (2) The initial grass amount is: (10 − 5) * 20 = 100. (3) Suppose 25 cows can eat for x days: (25 − 5) * x = 100, x = 5. Example 2: A piece of pasture has a certain stock of feed, and an equal amount of feed is purchased every day. Five sheep can eat the feed for 20 consecutive days, or six sheep can eat the feed for 15 consecutive days. If it is required to eat all the sheep in 6 days, how many sheep do you need at least? Answer: Suppose that each sheep eats 1 feed per day, (1) The rate of feed increase per day is: (5 * 20 − 6 * 15) / (20 − 15) = 2. (2) The original feed quantity is: (5 − 2) * 20 = 60. (3) How many sheep do you need at least? (x − 2) * 6 = 60, x = 12).2.1.2.1.The conceptual prior knowledge test consisted of three open-ended questions about the primary school math Olympiad problem (“cow eating grass”). Before the experiment, each participant was tested for the conceptual prior knowledge level to exclude the influence of the participants’ original knowledge and experience regarding the experiment.2.1.2.2.via E-prime.Explained the problem-solving ideas, principles, and corresponding formulas of the example (“cow eating grass” problem) [such as grass growth rate = (grazing speed 1 * time 1 − grazing speed 2 * time 2) / (time 1 − time 2)], presented separately 2.1.2.3.In the acquisition phase, regarding the restudy example condition, Examples 1 and 2 on the one hand, and Examples 3 and 4 on the other, contained the same problem-solving steps. In the condition of the example-problem pairs, Examples 1 and 3 were for learning in the form of examples, while Examples 2 and 4 were for solving problems. In the incomplete example condition, Examples 1 and 3 were for learning in the form of examples, while Examples 2 and 4 were for the supplementing solving steps.2.1.2.4.The retention test comprised two questions, one of which was similar to what appeared in the initial test task ; the other was different and would increase in difficulty, but both had come up before .2.1.2.5.very, very low effort to (9) very, very high effort, to examine the problem-solving skills learning of diverse participant groups at the level of mental effort invested.The participants were required to complete the Mental Effort Rating Scale after the acquisition phase and the retention tests. The scale was developed by 2.1.3.The experimental process was divided into the acquisition phase and the retention tests. Experiments were carried out in a quiet environment. Before the formal experiment, participants were asked to complete the conceptual prior knowledge test, and they were randomly assigned to each condition, then the computer presented the basic process and precautions of the experiment. In formal experiments, each group consisted of four similar examples. The computer presented each example individually for a maximum of 4 min per example.2.1.3.1.This phase was mainly for example study only. For the example study only, each participant group did not have to solve the problem alone, all the solution steps will be given, and the participants only needed to learn the steps. In the restudy examples condition, the participants continued to study the following example only after studying an example, until they finished four examples. For the example-problem pairs condition, after completing the example study only, the participants were presented with the problem of similar materials without a solution step, and required to answer on the answer sheet until they completed four examples. In the incomplete example condition, after completing the example study only (including the problem and specific problem-solving steps), the participants were presented with similar but incomplete examples (the problem-solving steps of the examples lacked the last step). The “incomplete problem” manipulation in Experiment 1 are shown below. At first, present an example. Then, the incomplete problem needed to be completed by the participants. There are three steps to solve the problem. Give the first two steps, and the third step needs to be completed by the participants themselves  The rate of feed increase per day is: (5 * 20 − 6 * 15) / (20 − 15) = 2. (2) The original feed quantity is: (5 − 2) * 20 = 60. (3) How many sheep do you need at least?). Participants complete the answers on the answer sheet until they completed four examples.2.1.3.2.The retention test had two questions, each was presented separately, and the participants needed to answer on the answer sheet. For the retention test answer score, each question consisted of three solution steps, three points for complete correctness, one and two points for partial correctness, and zero points otherwise. The points awarded based for the correct strategy use, considering the correct solution is reached for each step. As long as the strategy is correct, even if the result is wrong, points will be given. After completing the retention test, the participants in the immediate test were also required to perform a five-minute distraction task (watch a video); while those in the delayed test had to perform a distraction task. After a week, the participants on the delayed test took the retention test, while those on the immediate test completed a distraction task. The participants were required to rate their mental effort levels both during the study period and after the retention test.2.2.F = 0.104, p > 0.05. In terms of retention test results, the main effect of different initial test conditions was not significant, F = 0.493, p = 0.612, ηp2 = 0.008; that is, the difference in the scores of the three conditions of restudy example, example-problem pairs, and the incomplete example did not reach a significant level. Meanwhile, the main effect of different test times was also not significant, F = 0.042, p = 0.837, ηp2 = 0.000; that is, the participants in the immediate retention test and the delayed retention test were not significantly different. The interaction difference between the initial test conditions and test time did not reach a significant level, F = 0.332, p = 0.718, ηp2 = 0.006  = 2.28, p = 0.134, ηp2 = 0.019. The main effect of different initial test conditions was significant, F = 3.66, p = 0.029, ηp2 = 0.059. The results of the LSD multiple comparisons revealed that the difference between the incomplete example and the example-problem pairs reached a significant level. The interaction difference between the initial test conditions and test time did not reach a significant level, F = 2.888, p = 0.06, ηp2 = 0.048  was insignificant, whether it was the example-problem pairs or the incomplete example group. The results were consistent with previous findings .Moreover, the incomplete example group was added to the initial test difficulty of this experiment. The incomplete example group and the example-problem pair group belong to the two example-problem connection methods, and both belong to the problem-solving group (retrieve practice group). However, the former belongs to the hierarchical level task, while the latter belongs to the whole level task. While both belong to the problem-solving method, their difficulty in solving the problem is different—the former is weaker, and the latter is stronger. Incomplete examples require the learner to complete/supplement solving steps  or the different participant motivations.3.The purpose of Experiment 2 was to examine the effect of retrieval practice on problem-solving under different material conditions. It also explored possible differences in mental effort input under each condition.3.1.3.1.1.M = 19.98, SD = 3.45) who did not participate in similar experiments before. They were randomly assigned to one of the conditions. However, because some participants did not continue to participate in the delayed retention test, so there were only 111 participants left: different material example-problem pairs group (n = 18), the different materials restudy example group (n = 18), the same materials restudy example group (n = 16), similar materials example-problem pairs (n = 19), same materials example-problem pairs and similar materials restudy example groups (n = 20).The participants were 120 Minnan Normal University students ; “similar materials” refers to the same structure but different surface features; “different materials” refers to the variability of examples with dissimilar structures and surface features. It does not mean any example, but also belongs to the “cow eating grass” problem. The different examples are as follows: the Example 1 and 2. They have different structures and surface, but all belong to the “cow eating grass” problem. Example 1: A pasture is covered with grass, which grows at a uniform speed every day. This pasture can feed 10 cows for 20 days, or 15 cows for 10 days. How many days can 25 cows eat? Answer: suppose that the daily grass consumption of each cow is 1, (1) The daily growth rate of grass is: (10 * 20 − 15 * 10) / (20 − 10) = 5. (2) The initial grass amount is: (10 − 5) * 20 = 100. (3) Suppose 25 cows can eat for x days: (25 − 5) * x = 100, x = 5. Example 2: As the weather gets colder, the grass on the pasture decreases at a uniform speed every day. The grass can feed 20 cows for 5 days, or 16 cows for 6 days. How many days can 11 cows eat? Answer: suppose that the daily grass consumption of each cow is 1. (1) The rate of grass reduction per day is: (20 * 5 − 16 * 6) / (6 − 5) = 4. (2) The initial grass amount is: (20 + 4) * 5 = 120. (3) Suppose 11 cows can eat for x days? (11 + 4) * x = 120, x = 8. In the acquisition phase, regarding the restudy example condition, if the same material is used, then Examples 1 and 2, on the one hand, as well as Examples 3 and 4, on the other contain the same problem-solving steps. If similar or different materials were selected, Examples 1 and 2, on the one hand, as well as Examples 3 and 4, on the other, contained similar or different problem-solving steps. In the example-problem pairs condition, Examples 1 and 3 were for learning in the form of examples, while Examples 2 and 4 were for solving problems. The steps for solving the problem in Examples 2 and 4 are the same if the same materials were utilized as in Examples 1 and 3, respectively. If similar or different materials were selected, the problem-solving steps of Example 2 were similar or different from those of Example 1, and those of Example 4 were similar or different from those of Example 3.3.1.3.A mixed experimental design of 2  × 3  × 2  was adopted. The initial test conditions and materials were manipulated a between-subjects design, and the testing time was manipulated a within-subjects design. The dependent variables were the final test scores and mental effort scores.3.1.4.The procedure was identical to that used in Experiment 1, except the acquisition phase removed the incomplete example and learned materials with different properties. Acquisition phase: this phase was mainly to learn about similar, same, or different examples. For studying examples only, each group of participants did not have to solve the problem alone, all the solution steps would be given, and the participants only needed to learn the steps. For the same materials, in the restudying example condition, the participants continued to study the same examples after studying the example, until they had studied four examples; In the example-problem pair condition, after the example was studied, the problem of the same materials (with no solution steps) were presented, and the participants responded on the answer sheet until they were completed. The same process was followed for similar materials and different materials.3.2.F = 0.355, p > 0.05. Repeated measures analysis of variance was performed on initial learning conditions × material properties × testing time. In terms of final scores, the main effect of learning conditions was not significant, F = 0.735, p = 0.393, ηp2 = 0.007; the main effect of material properties was not significant, F = 2.13, p = 0.124, ηp2 = 0.038; the main effect of test time was significant, F = 5.24, p = 0.024, ηp2 = 0.047, that is, the participant’s performance on the immediate test was significantly lower than the delayed test; the interaction between learning conditions and test time did not reach a significant level, F = 1.378, p = 0.243, ηp2 = 0.013, the interaction difference between material properties and testing time did not reach a significant level, F = 0.532, p = 0.589, ηp2 = 0.01. The interaction between learning conditions and material properties did not reach a significant level, F = 1.442, p = 0.241, ηp2 = 0.027. The interaction of three variables did not reach a significant level, F = 1.953, p = 0.147, ηp2 = 0.036  = 0.003, p = 0.957, ηp2 = 0.00; the main effect of the material property was also not significant, F = 0.49, p = 0.614, ηp2 = 0.009; the main effect of test time was significant, F = 4.312, p = 0.04, ηp2 = 0.039, that is, the mental effort of the participants in the immediate test was significantly higher than that in the delayed test; the interaction difference between learning conditions and test time did not reach a significant level, F = 1.435, p = 0.234, ηp2 = 0.013; the interaction of material properties and test time did not differ significantly, F = 1.68, p = 0.191, ηp2 = 0.03. The interaction between learning conditions and material properties reach a significant level, F = 4.409, p = 0.015, ηp2 = 0.077. The similar materials reach a significant level, p = 0.021. The interaction of three variables did not reach a significant level, F = 1.055, p = 0.352, ηp2 = 0.02 .The final scores and mental effort input had significant main effects on test time. The immediate test scores were significantly lower than those under the delayed test, and the mental effort input in the immediate test was significantly higher than that under the delayed test. It indicates that the final scores and mental effort may be inversely proportional; that is, the higher the performance, the lower the mental effort input.In terms of difficulty, different materials were more challenging than similar materials, which were more difficult than the same materials. Theoretically, the same materials should have the best scores, and the different materials would be the worst; however, in some cases, particularly when restudying, the opposite may be true. In contrast to the example-problem pairs condition, the participants under the restudy example condition frequently overestimated their performance and did not engage in continuous study, which led to lower scores. However, due to its higher material difficulty, the restudy example under different materials may enhance the participants’ learning motivation.4.The purpose of Experiment 3 was to examine the effect of retrieval practice on acquiring problem-solving skills under different feedback times. As retrieving could be assumed to be more effortful than restudying, potential differences in the mental effort were explored.4.1.4.1.1.M = 20.98, SD = 3.45) who had not engaged in similar experiments before. They were randomly assigned to any of the four conditions (n = 20). As the participants under some conditions did not take part in the delayed test 1 week later, group size varied as follows: the immediate feedback example-problem pairs group (n = 12), the immediate feedback restudy examples group (n = 19), the delayed feedback example-problem pairs group (n = 14), the delayed feedback restudy examples group (n = 17).The participants were 80 Minnan Normal University students  * 2  * 2  was adopted. The initial test conditions and feedback time used a between-subjects design, and the testing time was manipulated a within-subjects design. The dependent variables were the final test scores and mental effort scores.4.1.4.The procedure was identical to that used in Experiment 1, with the following exception: removed the incomplete example condition and added feedback  during the acquisition phase.Acquisition phase: This stage was mainly to learn from similar examples. For study only, the participants in each group did not have to try to solve the problem by themselves, all the solution steps were given, and they merely had to learn the steps. In the example-problem pair condition, if immediate feedback was given, after the participants had answered Examples 2 and 4, they presented all the steps to solve the problem, respectively; that is, the material presentation method was Example 1 − Example 2 − Feedback (The problem-solving steps of Example 2) − Example 3 − Example 4 − Feedback (The problem-solving steps of Example 4) until four examples were completed. The presentation time of feedback (problem-solving steps) was 1 min. Then they took the immediate test and a delayed test a week later. If delayed feedback was given, it was after completing the four examples, taking part in the instant test, and giving feedback a week later. This process occurred when the problem-solving steps of Examples 2 and 4 were presented completely, and the presentation time was 1 min, respectively, and followed by the delayed test. In the restudy examples condition, the participants continued to study similar examples after studying an example, until they finished four examples; if immediate feedback was given, to maintain balance, the participants only study were extended the study time. The total time was the same for both study conditions. Then they would take the immediate test and a delayed test a week later. If delayed feedback was given, it was after completing four examples, participating in the immediate test, and receiving feedback a week later. They then restudied Examples 2 and 4, and the presentation time was 1 min, respectively, and subsequently, they took part in the delayed test again.4.2.F = 0.075, p > 0.05. An ANOVA was performed on the learning conditions, feedback, and test time. In terms of the final score, the main effect of the learning conditions was not significant, F = 0.406, p = 0.527, ηp2 = 0.007; the main effect of feedback was not significant, F  = 0.31, p = 0.58, ηp2 = 0.005; the main effect of test time was also not significant, F = 1.725, p = 0.194, ηp2 = 0.028; the interaction difference between learning conditions and test time was not significant, F = 0.976, p = 0.327, ηp2 = 0.016; the interaction difference between the feedback and test time was not significant, F = 2.967, p = 0.09, ηp2 = 0.048. The interaction between the learning conditions and the feedback did not reach a significant level, F = 0.307, p = 0.582, ηp2 = 0.005. The interaction of three variables did not reach a significant level, F = 3.964, p = 0.051, ηp2 = 0.064  = 0.071, p = 0.0791, ηp2 = 0.001; the main effect of feedback was also not significant, F = 0.023, p = 0.879, ηp2 = 0.000; the main effect of test time not significant, F = 1.489, p = 0.227, ηp2 = 0.025; the interaction between learning conditions and test time did not reach a significant level, F = 0.573, p = 0.452, ηp2 = 0.01; the interaction between the feedback and the test time was not significant, F = 1.345, p = 0.251, ηp2 = 0.022. The interaction between the learning conditions and the feedback did not reach a significant level, F = 0.043, p = 0.836, ηp2 = 0.001. The interaction of three variables did not reach a significant level, F = 0.27, p = 0.606, ηp2 = 0.005  was more inclined to have its characteristics. The example effect of this knowledge system is structured, non-declarative knowledge , and thus, creates the opposite result of the retrieval practice effect (under the condition of delayed feedback). However, it did not occur under the condition of immediate feedback, which may be because the difficulty of retrieval of immediate feedback is lower than that of delayed feedback. Furthermore, the experiment used similar materials, and the combination of the two feedback types did not generate the effect of retrieval practice.Studies have shown that feedback contributes to the retrieval practice effect . For tho5.The three experiments delineated above jointly find that the retrieval practice effect may not apply to acquiring problem-solving skills. The restudy examples only were used for repeat studying. The example-problem pairs with only questions and no problem-solving step results were used as retrieval practice, that is, the participants were asked to retrieve content. This study was mainly carried out by retrieving three aspects of the difficulty of practice, namely, initial test difficulty, material difficulty, and feedback difficulty. The difficulty of the initial test was divided into learning conditions: restudy example, example-problem pairs, and incomplete example. The difficulty of materials was divided according to the properties of materials: similar, same, and different. The difficulty of feedback was divided based on timing: immediate feedback (after 5 min) and delayed feedback (after 1 week). Through a step-by-step experiment, it was found that the retrieval practice effect cannot be realized in the example.As the students were primarily in the cognitive framework or mode of the learning step procedure during the study of the example, they appeared to benefit less from the learning effects of problem-solving. Recent studies have shown that the combination of studying examples and problem-solving yields little benefit . Baars eRegarding the mental effort input, Experiment 1 demonstrated that under the immediate test, the example-problem pairs group was more invested than the incomplete examples group, and the difference was significant. Studies have established that the effort value of example-problem pairs will be more than that of restudy examples . HoweverRestudying/feedback after testing, according to The failure of the retrieval practice effect on the example study may be relatively due to the short acquisition phase . The parProvided the critical prompt in the acquisition phase, retrieval practice enhanced analogical problem-solving . HoweverThe key takeaway from our study is that procedural knowledge, one component of skill knowledge acquisition, produces the least significant effects. According to current research, this type of information, which is more closely related to working memory than declarative knowledge, was highly correlated with context or structure. However, neither the division of procedural knowledge nor the division of experimental materials has an operational definition. There are no quantitative measures, even though The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Academic Committee of Minnan Normal University. The patients/participants provided their written informed consent to participate in this study.XH conceived and designed this study, collected and analyzed the data, and wrote and revised the manuscript. SZ, ZY, and SC revised the manuscript. XH and SC were responsible for the study. All authors contributed to the article and approved the submitted version.This research was supported by the National Office for Education Sciences Planning, Ministry of Education of the People’s Republic of China through Grant BBA220203.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


N6-methyladenosine (m6A) modification regulators are essential for the diagnosis and treatment of various cancers. However, the comprehensive analysis about roles of m6A “readers” in hepatocellular carcinoma (HCC) remains unclear. UALCAN, GEPIA2, HPA, Kaplan Meier plotter, cBioPortal, STRING WebGestalt, Metascape and TIMER 2.0 database and Cytoscape software were used to comprehensively analyze the bioinformatic data. We found that m6A “readers” were upregulated at the mRNA level and protein level in HCC patients. Highly expressed YTHDF1, IGF2BP3 and NKAP were positively correlated with advanced HCC stage and had a poor prognosis in OS and PFS. The gene alterations of m6A “readers” happened frequently, and YTHDF3 had the highest mutation rate. The function of m6A “readers” on HCC may be closely correlated with splicing related proteins , metabolic process, protein binding and RNA splicing related signaling pathways. Moreover, although the correlation of YTHDF3 and CD8+ T cell infiltration, and the correlation of IGF2BP3 and infiltration of mast cells and CAF are negative, most m6A “readers” had a positive correlation with immune cells . Macrophages, CD4+ T cell, Treg, B cell, monocyte, and myeloid dendritic cell had a positively strong correlation (Rho>0.4) with most m6A “readers” . In conclusion, by comprehensive analysis of m6A “readers”, we found that they were involved in the prognosis of HCC, and m6A “readers” might regulate the development and progression of HCC by participating in some metabolism-related and RNA splicing-related signaling pathways as well as immune cell infiltration. Primary liver cancer was reported as the sixth most commonly diagnosed cancer as well as the third leading cause of cancer death worldwide in 2020. Statistical data shows that it was about 906,000 new cases and 830,000 deaths. And liver hepatocellular carcinoma (HCC) accounts for 75% to 85% of primary liver cancer . With thN6-methyladenosine (m6A) modification has been a research hot spot in recent years. m6A modification is the most abundant modification in eukaryotic RNA, with roles including maintenance of RNA stability, mRNA precursor splicing, translation initiation, nuclear export, degradation, etc. . It was In this paper, we sort out a comprehensive bioinformatics analysis of the m6A “readers”  in HCC. In addition, we discussed their values to be potential therapeutic targets and prognostic biomarkers through various public bioinformatics databases. Furthermore, we selected some key genes from the co-expression genes of m6A “readers”. And these key genes as well as some immune cells infiltration may provide may provide new ideas for the subsequent study of the mechanism of m6A modification in HCC.We used UALCAN database to check the mRNA expression level of m6A “readers” between HCC and normal liver tissue. The expression level of m6A “readers” such as YTHDC1, YTHDC2, YTHDF1, YTHDF2, YTHDF3, IGF2BP1, IGF2BP2, IGF2BP3, HNRNPA2B1, HNRNPC, and NKAP were explored. To our surprise, all of these “readers” expressed significantly higher in HCC than that in normal tissues . ImmunohUALCAN was used to evaluate the association between the mRNA expression level of the m6A “readers” and the pathological stages of HCC patients . we usedNext, we analyzed the prognostic values of mRNA expression level of m6A “readers” in HCC patients through Kaplan-Meier plotter. It was obvious that HCC patients with higher mRNA levels of YTHDF1, YTHDF2, IGF2BP1, IGF2BP2, IGF2BP3, HNRNPC, NKAP displayed shorter overall survival (OS) time . The mRNTo further analyze the functions of m6A “readers” in HCC patients, we focused on the alteration profiles of m6A “readers”. cBioPortal database was used to evaluate the genetic alterations of the m6A “readers” . AccordiTo know the regulatory mechanisms of m6A “readers” in HCC, we picked out the top one co-expression genes of m6A “readers” . As is sFunctional enrichment analysis allows hundreds of genes to be allocated in different pathways thus contributing to the understanding of an unknown gene. In this study, we used the WebGestalt database to evaluate the biological functions of the m6A “readers” from pan-cancer analysis.From the GO analysis, it was clearly that the most highly enriched biological process (BP) category was metabolic process, followed by biological regulation, cellular component organization and response to stimulus. In the cellular component (CC) categories, nucleus, membrane-enclosed lumen, protein-containing complex, membrane, and cytosol were highly enriched. As for the molecular function (MF) category, the co-expression genes of m6A “readers” were mainly enriched in protein binding, nucleic acid binding, and ion binding .In addition, from the KEGG pathway results , it was In addition, we integrated data from Kaplan-Meier plotter database, UALCAN database and GEPIA2 database, and it was not difficult to find that YTHDF1, IGF2BP3 and NKAP were involved in OS, PFS, and stage in HCC . So, we To explore the correlation between the m6A “readers” and immune cell infiltration in HCC, we searched some information from the TIMER 2.0 database , 4. As iTo verify the reliability of the results, we carried out further studies using IGF2BP3 as a representative gene . First, we used immunofluorescence results from HPA database and demonstrated that IGF2BP3 was mainly localized in the cytoplasm . IGF2BP3Based on the expression level of IGF2BP3 from CCLE database, we stably knocked down the IGF2BP3 mRNA level in Hep3B cell, and the knocked-down (KD) efficiency were checked by RT-PCR  and WestThe biological importance of m6A modifications mainly depends on the roles of m6A “readers” which control RNA fate and function. Studies showed that some m6A “readers” were involved in the development of HCC. For instance, Tanabe A and his colleague proved that through activation of c-Jun and ATF-2, YTHDC2 played a significant role in promoting HCC cell growth . In addiIn this study, we conducted a comprehensive study on the expression level and clinical prognostic value of m6A “readers” in HCC. We showed the different expression of m6A “readers” between HCC and normal tissues in mRNA and protein level. Our results showed that the mRNA expression level of the common m6A “readers” including YTHDC1, YTHDC2, YTHDF1, YTHDF2, YTHDF3, IGF2BP1, IGF2BP2, IGF2BP3, HNRNPA2B1, HNRNPC, and NKAP were highly expressed in HCC. And the protein expression level of most of m6A “readers” was the same as mRNA expression. In addition, YTHDC1, YTHDF1, YTHDF2, IGF2BP3, HNRNPA2B1 and NKAP were confirmed by two databases to be positively correlated with tumor stage, which deserved more attention in the treatment of HCC. Besides, our results implied that YTHDC1, YTHDF1, YTHDF2, IGF2BP1, IGF2BP2, IGF2BP3, HNRNPA2B1, HNRNPC, and NKAP had a prognosis value in either OS or PFS. But only YTHDF1, IGF2BP2, IGF2BP3 and NKAP had significant value to predict both OS and PFS time in HCC patients. This seems a little inconsistent with the previous results . Due to Somatic mutation and clonal selection were two major reasons for cancer development , 24. TheThe co-expression genes may give some new thoughts to the development and progression of HCC. For instance, they may be potential targets of m6A “readers”, or be regulators of m6A “readers”. To determine the functions of these m6A “readers” in HCC, we depicted a PPI network based on the co-expression genes of m6A “readers”. The top 10 hub genes seem from three families . In addition, HNRNP, SNRP, and SR proteins were all discovered as regulators of alternative splicing. HNRNP family was a large family of RNA-binding proteins (RBPs) that participated in multiple aspects of nucleic acid metabolism, which including alternative splicing, mRNA stabilization, transcriptional regulation and translational regulation . Small nThe GO and KEGG enrichment analysis helped us to understand the functional meanings of genes at a molecular level , 37. AndIn recent years, immunotherapy becomes a popular research field. It is noteworthy that clinically detected cancers must evade the anti-tumor immune response before they can grow gradually. Tumor immune monitoring is essential for the body to eliminate tumor cells. Studies have shown that tumors can resist immune attack through immune system suppression pathways and immune system exclusion or neglect . The tumWe found that the expression of the m6A “readers” is significantly positively linked to the infiltration of CD8+ T cell, CD4+ T cell, Tregs, B cell, neutrophil, monocyte, macrophage, DC, NK cell, and mast cell. It was worthy to pay attention to the correlation between HNRNPC and DC. They had a high correlation value 0.535. In addition, among these immune cells, macrophage might play a core role in m6A “readers”, because it showed positively strong correlation with most m6A “readers”. Macrophage was reported as potent immune effector cells, and it had a functional plasticity which contributed to both antitumor and protumor function in different background . In thisIn further analysis and validation of IGF2BP3 functions in cell model, we found that IGF2BP3 showed high expression in HCC cell lines according to the CCLE database, and knockdown of IGF2BP3 inhibited the migration and sphere formation ability of HCC cells. These results suggested that IGF2BP3 might act as an oncogene and was related to the poor prognosis of HCC patients by enhancing cell migration and stemness ability.In this study, we systematically collated the expression data of the common m6A “readers” in HCC, and analyzed their clinical value. In addition, we speculated their possible functions and discussed their correlation with immune cell infiltration. It was worthy to note that YTHDF1, IGF2BP3 and NKAP may be potential therapeutic targets and biomarkers. Because they were expressed differently, and were negatively correlated to OS, PFS, and tumor stage in HCC. More importantly, YTHDF1, IGF2BP3 and NKAP were also highly expressed in pan-cancer, which provided the promise of YTHDF1, IGF2BP3 and NKAP as potential therapeutic targets. From the results of the functional enrichment analysis, the main functions of these three potential therapeutic targets are involved in the regulation of cell cycle or protein localization. In addition, the functions of m6A “readers” in HCC may be related to splicing proteins such as HNRNP, SNRP and SR. And the immune cells especially the macrophage, CD4+ T cell, Treg, B cell, monocyte, and myeloid dendritic cell may play a significant role in the progression of HCC. Besides, immune microenvironment of m6A “readers” was also worthy to be explored. In conclusion, our findings in this study could give some new idea to the development of multiple molecular diagnostic biomarkers and treatment targets for HCC patients to improve their prognosis.Protein expression patterns in cancers contain crucial diagnostic and prognostic information. However, from the HPA database, we could not obtain the protein expression information of YTHDF1 and YTHDF3 in HCC. In addition, there was no data to present the correlations between m6A “readers” and metastasis of multiple lymph node in HCC, and it may be caused by insufficient sample size or other reasons. Besides, we found that most of the m6A readers were associated with the prognosis of HCC patients, and the m6A “readers” were found to be closely related to the immune microenvironment. So, in the near future, we will compose a prognostic model incorporating the m6A “readers”  and immune microenvironment components  to better complement this study. Furthermore, our research was mainly based on public databases, and the results of this study should be further validated by more laboratory experiments.Multiple bioinformatics database resources were accessed to explore the expression levels of m6A “readers” in HCC samples. The website tools  were based on the TCGA database, which contains 371 HCC samples and 50 normal liver samples. And TPM (Transcripts per million) data was applied for the mRNA expression level analysis.http://ualcan.path.uab.edu/index.html. We used UALCAN database to evaluate the expression difference between HCC and normal tissue. And the differences in transcriptional expression were compared by Student’s t-test, we considered P < 0.05 as statistically significant. In addition, the figures of pathological stage and lymph nodes metastasis were also derived from this database.The UALCAN database is an interactive portal for in-depth analysis of gene expression data from the TCGA database. It compares the tumor and normal tissue samples based on pathological stage, lymph nodes metastasis and other clinicopathological characteristics . UALCAN http://www.proteinatlas.org. In this study, we used the HPA database to show the different expression of m6A “readers” between HCC and normal tissues.The Human Protein Atlas database is an immunohistochemistry-based map of protein expression profiles in normal tissues, cancers and cell lines. Proteome analysis was based on 26941 antibodies targeting 17165 unique proteins. It allows complex queries , so as to provide a resource for pathology-based research . And thehttp://gepia2.cancer-pku.cn/. In this study, we used GEPIA2 database to show the correlation between m6A “readers” and HCC stage.GEPIA2 was available to analyze gene expression quantification both in the gene level and the transcript level. In addition, it was able to analyze and compare different cancer subtype . GEPIA2 http://kmplot.com/analysis/. We used Kaplan-Meier plotter to analyze the prognostic value including overall survival (OS) and progression free survival (PFS) of the m6A “readers” in HCC patients. We divided 371 HCC specimens into high and low expression groups according to their mRNA expression by automatically selecting the best cut-off value. Kaplan-Meier analysis was evaluated by log-rank test, and we considered P < 0.05 as statistically significant difference.The Kaplan Meier plotter could evaluate the effect of 54k genes  on survival in 21 cancer types. The correlations between gene expression and survival were computed using the Cox proportional hazards regression. And the sources for the databases contain GEO, EGA, and TCGA . Kaplan-https://www.cbioportal.org. In this study, we used cBioPortal to analyze the genome map of the m6A “readers” in HCC and obtain the mutation and mRNA expression data. Besides, we downloaded the co-expression genes of m6A “readers” from cBioPortal for further analysis.cBioPortal database provides a Web resource for exploring, visualizing, and analyzing cancer genomics data. It provides and simplifies the summaries of gene-level data from multiple platforms . cBioPorhttps://string-db.org/. In our study, STRING was applied to explore and analyze possible protein-protein interactions (PPIs).The STRING database collects and integrates publicly available sources of protein-protein interaction information, and provides the computational predictions. And it aims to achieve a comprehensive and objective global network among proteins . STRING Cytoscape is a popular tool for analyzing and visualizing multiple proteins or genes and for integrating biomolecular interaction networks into a unified conceptual framework . In our http://www.webgestalt.org/. In our study, we used WebGestalt to provide enrichment analysis results including Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of co-expression genes of m6A “readers”.WebGestalt is a tool for the interpretation of gene lists. It supports three well-established and complementary methods for enrichment analysis, including over-representation analysis (ORA), gene set enrichment analysis (GSEA), and network topology-based analysis (NTA) . WebGesthttps://metascape.org/gp. In this study, we used Metascape to analyze the functional enrichment of m6A “readers” and their co-expression genes.Metascape, a gene annotation and analysis resource, integrated functional enrichment, interactome analysis, gene annotation, and membership together . Metascahttp://timer.cistrome.org/. In this study, we used TIMER2.0 to depict the relationship between m6A “readers” and immune cells including CD8+ T cell, CD4+ T cell, T cell regulatory (Tregs), B cell, neutrophil, monocyte, macrophage, myeloid dendritic cell (DC), nature killer (NK) cell, mast cell, and cancer associated fibroblast (CAF). All of these data had a purity adjustment process. And log2 RSEM was used for displaying the gene expression levels.TIMER is a comprehensive resource for the analysis of immune infiltrates among various cancer types. TIMER2.0 provides immune infiltrates’ abundances estimated by multiple immune deconvolution methods . TIMER2.https://portals.broadinstitute.org/ccle).Cancer Cell Line Encyclopedia (CCLE) database can be used to explore gene variants, candidate targets, small molecules and biotherapeutics, and characterize cancer cell lines . In this2.Hep3B cells were purchased from Zhong Qiao Xin Zhou Biotechnology Co., Ltd.  and were cultured in MEM medium  containing 10% fetal bovine serum (FBS) , 100 μg/mL streptomycin and 100 IU/mL penicillin . They were cultured at 37°C, 5% COStable KD of IGF2BP3 was obtained by a lentiviral-based shRNA system. Target specific shRNAs or nontarget control (NC) were subcloned into GV493(hU6-MCS-CBh-gcGFP-IRES-puromycin) vector. To isolate infected Hep3B cells, Puromycin selection was performed at 2 ug/ml for 7 days. The KD efficiencies were verified at both mRNA and protein levels. The shRNA sequences used in this study are as follows. shIGF2BP3#1, 5′-ATAGGTTACATTTACAACTGC-3′, shIGF2BP3#2, 5′-TAATCCAGGAATTAAATGTGC-3′, shNC, 5′-TTCTCCGAACGTGTCACGT-3′. 1 μg/ml puromycin for selection was added to the medium of cells transduced with lentivirus.−ΔΔCt methods relative to GAPDH. Specific primer sequences were as follows: IGF2BP3: 5′-ACGAAATATCCCGCCTCATTTAC-3′ (forward), 5′-GCAGTTTCCGAGTCAGTGTTCA-3′ (reverse); GAPDH: 5′-CTGGGCTACACTGAGCACC-3′ (forward), 5′-AAGTGGTCGTTGAGGGCAATG-3′ (reverse).Total RNA was extracted from Hep3B cells which transfected with shNC and shIGF2BP3 lentivirus, according to the RNA Isolater . The concentration and purity of total RNA were measured. Through SureScript First-strand cDNA synthesis kit , total RNA was used to obtain cDNA by reverse transcription reaction. The RT-PCR assays were carried out by a protocol from Power SYBR Green . The relative expressions of genes were calculated and normalized using the 2Whole cell lysates were collected in RIPA lysis buffer . The antibodies used in this study are IGF2BP3 , and β-actin  antibodies. HRP-conjugated anti-rabbit secondary antibody was used for the ECL detection.5 cells/ml and 200 μl of the suspension was inoculated in transwell chambers. After 48 hrs of incubation, the cells were fixed in methanol for 30 mins and stained with crystal violet for 30 mins.800 μl of conditioned medium containing 20% fetal bovine serum was used as a chemotactic agent for the bottom chamber of the 24-well plate. Serum-free medium of cells transfected with lentivirus shNC and shIGF2BP3 were prepared as suspension at 5 × 10Lentiviral transfected shNC and shIGF2BP3 cells were digested with trypsin into single cell suspensions and seeded into special 24-well plates (with 1% polyHEMA (Sigma)-coated) at a density of 3000 cells per well. Cells were grown for 10–14 days in special medium  supplemented with 4mg/mL insulin , B27 , 20 ng/mL EGF , and 20 ng/mL FGF . The numbers of tumor spheres larger than 50 μm in diameter were calculated.t-tests, while differences among more than two groups were analyzed by ANOVA. The two patient cohorts are compared by a Kaplan-Meier survival plot, and the hazard ratio with 95% confidence intervals and log-rank P value are calculated. Pearson and Spearman correlation analysis were used to analyze correlations of the data. The P < 0.05 was considered as statistical significance.The differences between two groups were analyzed by Student’s All methods were performed in accordance with the relevant guidelines and regulations.All data generated or analyzed during this study are included in this published article.Supplementary FiguresSupplementary Table 1