Prognostic Significance of MicroRNAs in Medulloblastoma: A Systematic Review and Meta-Analysis

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Introduction
Medulloblastoma (MB) is one of the most malignant neuroepithelial tumors of the central nervous system in children [1]. According to current consensus, MB has four core molecular subsets: WNT, SHH, Group 3and Group 4 [2], which differ not only distinct in their underlying genetic changes but also differ in clinical characteristics like age, gender-related incidence, the incidence of metastasis, and overall survival rates [3,4]. Standard treatment for MB includes surgery, adjuvant chemotherapy, and craniospinal irradiation; aggressive interventions lead to prolonged sequelae and poor quality of life [5]. Approximately 75% of pediatric MB patient's recurrence within a few years [6]. Therefore, the treatment of MB remains a challenge in pediatric oncology. Baliga S et al. [7] Studies indicate that the 10-year OS for standard and intermediate/high-risk patients was 86.9% and 68.9%, respectively. Despite progress in the treatments, approximately 30% of MB children will die from current treatment strategies [8], so reducing mortality and morbidity is urgent.
MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression at the post-transcriptional level [9]. MiRNAs bind to complementary sequences in the 3′ untranslated areas of numerous target genes, usually leading to their silencing [10]. Each miRNA is thought to target hundreds of genes. It has been revealed as a critical regulator during normal tissues and cancers [11,12]. More evidence indicates that miRNA expression disorder plays a vital role in pathogenesis，cancer progression and response to treatment [13]. Consequently, they have been extensively studied as diagnostic and prognostic cancer biomarkers in recent years [14,15]. These efforts aim to find new molecular markers and targeted therapies to achieve early diagnosis and better treatment. MiRNAs are associated with prognosis in patients with MB, but the conclusions have been inconsistent. A previous systematic review explored the relationship between miRNA and prediction in MB patients. However, only two studies were included, thus lacking relevant data. The survival outcome of tumor patients was not quantitatively analyzed, and miRNA was not found to have guiding significance for the prognosis of pediatric MB patients. This systematic review and meta-analysis aim to clarify the relationship between miRNA and the prognosis of patients with MB through a systematic and comprehensive qualitative and quantitative analysis of existing original studies.

Guidelines and Registration
The Preferred Reporting Items for Systematic Review and Meta-analysis (PRISMA) criteria were used to perform the study [16]. This study is based on the PROSPERO that was registered under the ID CRD42021289410. Selection process 8

Item
Specify the methods used to decide whether a study met the inclusion criteria of the review, including how many reviewers screened each record and each report retrieved, whether they worked independently, and if applicable, details of automation tools used in the process.

6-7
Data collection process 9 Specify the methods used to collect data from reports, including how many reviewers collected data from each report, whether they worked independently, any processes for obtaining or confirming data from study investigators, and if applicable, details of automation tools used in the process.

6-7
Data items 10a List and define all outcomes for which data were sought. Specify whether all results that were compatible with each outcome domain in each study were sought (e.g. for all measures, time points, analyses), and if not, the methods used to decide which results to collect.

10b
List and define all other variables for which data were sought (e.g. participant and intervention characteristics, funding sources). Describe any assumptions made about any missing or unclear information. 7 Study risk of bias assessment 11 Specify the methods used to assess risk of bias in the included studies, including details of the tool(s) used, how many reviewers assessed each study and whether they worked independently, and if applicable, details of automation tools used in the process.

7
Effect measures 12 Specify for each outcome the effect measure(s) (e.g. risk ratio, mean difference) used in the synthesis or presentation of results. 7

Synthesis methods
13a Describe the processes used to decide which studies were eligible for each synthesis (e.g. tabulating the study intervention characteristics and comparing against the planned groups for each synthesis (item #5)).

13b
Describe any methods required to prepare the data for presentation or synthesis, such as handling of missing summary statistics, or data conversions. 8 13c Describe any methods used to tabulate or visually display results of individual studies and syntheses. 8 13d Describe any methods used to synthesize results and provide a rationale for the choice(s). If meta-analysis was performed, describe the model(s), method(s) to identify the presence and extent of statistical heterogeneity, and software package(s) used.

13e
Describe any methods used to explore possible causes of heterogeneity among study results (e.g. subgroup analysis, meta-regression). 8 13f Describe any sensitivity analyses conducted to assess robustness of the synthesized results. 8 Reporting bias assessment 14 Describe any methods used to assess risk of bias due to missing results in a synthesis (arising from reporting biases). 8 Certainty assessment 15 Describe any methods used to assess certainty (or confidence) in the body of evidence for an outcome. 8

Study selection
16a Describe the results of the search and selection process, from the number of records identified in the search to the number of studies included in the review, ideally using a flow diagram.

16b
Cite studies that might appear to meet the inclusion criteria, but which were excluded, and explain why they were excluded. 8

Study characteristics 17
Cite each included study and present its characteristics. 8-10 For all outcomes, present, for each study: (a) summary statistics for each group (where appropriate) and (b) an effect estimate and its precision (e.g. confidence/credible interval), ideally using structured tables or plots. 8

Results of syntheses 20a
For each synthesis, briefly summarize the characteristics and risk of bias among contributing studies. 9-10 20b Present results of all statistical syntheses conducted. If meta-analysis was done, present for each the summary estimate and its precision (e.g. confidence/credible interval) and measures of statistical heterogeneity. If comparing groups, describe the direction of the effect.

9-10
20c Present results of all investigations of possible causes of heterogeneity among study results. 9-10 20d Present results of all sensitivity analyses conducted to assess the robustness of the synthesized results.

9-10
Reporting biases 21 Present assessments of risk of bias due to missing results (arising from reporting biases) for each synthesis assessed. 11 Certainty of evidence 22 Present assessments of certainty (or confidence) in the body of evidence for each outcome assessed. 11

Discussion
23a Provide a general interpretation of the results in the context of other evidence. 12-14 23b Discuss any limitations of the evidence included in the review.

12-14
23c Discuss any limitations of the review processes used.

12-14
23d Discuss implications of the results for practice, policy, and future research.

Registration and protocol 24a
Provide registration information for the review, including register name and registration number, or state that the review was not registered. 5 24b Indicate where the review protocol can be accessed, or state that a protocol was not prepared. 5 24c Describe and explain any amendments to information provided at registration or in the protocol.

Inclusion Criteria
• Full-text articles in English; including patients with histopathologically diagnosed MB.
• Measured the expression of miRNAs in tumor tissue, serum, or plasma, as well as the survival prognosis of patients.
• Reported the survival curves for overall survival (OS) or disease-free survival (DFS) or cause-specific survival (CSS) or recurrence-free survival (RFS) with or without the hazard ratio (HR) and its 95% confidence intervals (CIs).

Exclusion Criteria
• Lack of patient survival data.
• Studies that included non-human data.
• Reviews, preclinical studies, and duplicate reports were excluded.

Data Extraction and Quality Assessment
Two authors independently performed database search, data extraction, and quality evaluation. The discussion will resolve any disagreement until consensus is reached or consulting a third author. Including miRNAs studied, first author's names, publication year, study location, platform, source of a clinical sample，sample size, study design, metastasis or not, result, survival data (HR and 95%CI), and Newcastle-Ottawa Scale (NOS). If HRs and 95% CIs were not provided directly in the included articles, we estimated them from Kaplan-Meier survival curves with methods described by Tierney et al. using Engauge Digitizer version 4.1 [17]. Two reviewers systematically evaluated the quality of included studies according to NOS. The higher the score, the better the quality of the essay. A joint review solved disagreement.

Statistical Analysis
Meta-analyses were performed to summarize the association between miRNA expression and OS in MB patients. HR and the corresponding standard errors were estimated from 95% CIs or p-values and were logarithmically transformed to obtain a normal distribution. The Cochrane's Q test and I ² test were performed to evaluate the heterogeneity [18]; an I ² > 50% indicates significant heterogeneity. A random-effect model was applied if substantial heterogeneity was detected; otherwise, a fixed-effect model was applied. Egger's test and funnel plot symmetry were used to assess the risk of publication bias. Sensitivity analyses were performed to evaluate the stability of the results by omitting each of the included studies one at a time [19]. The STATA software (Version 16.0; Stata Corporation, TX, USA) was used for the statistical analyses.

Study Selection
As shown in (Figure 1), the initial search resulted in 152 articles from PubMed (n = 38) and the Web of Science (n = 27), Embase (n = 87). After excluding, studies unrelated to our systematic review and meta-analysis according to the exclusion criteria, 22 articles were considered to be screened. The full text of the 22 articles was reviewed, and 8 of them did not measure miRNAs expression, non-human sample studies (n = 2), and the full text of the studies could not be obtained (n = 2). Full-text studies of qualitative synthesis according to inclusion criteria (n = 10), 4 of which lacked relevant data. Finally, 10 studies were included for systematic analysis, of which 6 papers could be further meta-analyzed.

Study Characteristics
The characteristics of the included studies are shown in (Table 1). Overall, the meta-analysis included 855 MB patients from 6 studies in China, Switzerland, Brazil, and India. All articles are published in English. Since the 2 studies included 2 independent miRNAs, respectively, and 1 study included 3 independent miRNAs, the relationship between miRNA expression in tumors and OS prognosis during follow-up was assessed in the article, so these were included independently. Finally, 10 miRNAs from 6 studies were included in the meta-analysis. The samples included ranged from 32 to 470. 6 miRNAs in the 4 studies included 4 molecular subsets in MB patients. In comparison, 4 miRNAs in the other 2 studies included Group 3 and Group 4 patients in the molecular subsets of MB. No cutoff value was mentioned in the 2 studies, and the median miRNA expression value was used as a cutoff value in the remaining 4 studies. 5 studies only used QRT-PCR for miRNA quantification, and 2 studies used both QRT-PCR and microarray analysis, and the outcome of OS was reported during follow-up. The NOS ranged from 6 to 8 in the included studies.

MicroRNAs First author
Year

Comprehensive Meta-Analysis
In 855 MB patients from 6 included studies, the prognostic significance of 10 miRNAs was investigated ( Figure 2). 7 miRNAs were upregulated, while 3 miRNAs were downregulated. The overall pooled effect estimates of HR for (upregulated and down-regulated) miRNA expressions were 0.57, with a 95 percent CI of 0.33 -0.99, meaning that miRNAs expression reduced the risk of death in MB patients when using a fixed-effect model.

Publication Bias
In the pooled analysis, funnel plot symmetry was used to assess the risk of publication bias. The funnel plot of the overall study is shown in Figure 4. The funnel plot symmetry was confirmed by visual examination, suggesting that the potential risk of publication bias was negligible. The result of Egger's test for this meta-analysis also demonstrated no significant publication bias. Results show that no significant publication bias was found in the analysis.

Sensitivity Analysis
Sensitivity analysis was performed by removing individual studies in turn. Our results were unchanged, indicating that our combined HRs and 95%CIs were stable in this meta-analysis. The result is shown in (Figure 5).

Figure 5:
Forest plot in the meta-analysis random-effects estimates (exponential form) sensitivity analysis by omitting one study by turns.

Discussion
The prognostic efficacy of miRNAs in various tumors has been studied, including breast cancer, gastric cancer, glioma, lung cancer and other cancers [20][21][22][23]. MiRNA, as a short non-coding RNA, regulates gene expression at the post-transcriptional level, especially playing a crucial role in the occurrence and development of human cancer [24]. Abnormal regulation of miRNA results in changes in downstream oncogenes, related cancer suppressors, and signaling pathway molecules [25]. Some patients with MB experience local or metastatic relapses after standard therapy, a condition associated with very poor prognosis [26]. Though the prognostic relationship between miRNA-182 [27], 204 [28], 100 [29], 137 [30] and other miRNAs and MB patients has been studied, the results are inconsistent. Previous systematic reviews and meta-analyses have also explored the potential prognostic role of miRNAs in many other cancers. Still, no quantitative meta-analysis has been studied to investigate the prophetic role of miRNA in MB patients [31][32][33]. Our meta-analysis is novel and examines miRNAs' prognostic meaning as biomarkers in MB patients using a continuous version pooled meta-analysis. The primary purpose is to study the relationship between miRNAs and the prognosis of MB patients and provide further theoretical evidence for clinical application.
Our meta-analysis investigated 855 MB patients with 10 miRNAs from 6 studies. The HR combined effect of related miRNAs expression (up-regulation and down-regulation) was estimated to be 0.57 with a 95% CI 0.33-0.99. These results suggested that the overall pooled effect of miRNA expression related to the prognosis of MB update could reduce the risk of death in MB patients. In addition, Subgroup analyses showed that prognostic efficacy of tumor miRNA level for better OS was stronger in MB patients of the four molecular subsets (HR：0.37; 95% CI：0.19-0.73 ；P=0.670) than those with Group 3 and Group 4 molecular subsets only (HR：0.79; 95% CI：0.29-2.16；P=0.024). The results are consistent with previous studies [34][35][36]. they have observed how miRNA expression differs depending on molecular subsets and metastasis factors. Both univariate and multivariate analyses were used to explore the survival rate of MB patients. The results indicate that dysregulation miRNA expression was associated with poor prognosis in MB with the Group3 and Group4 molecular subsets or in patients with metastases，suggesting that molecular subsets and metastasis may be independent prognostic factors of MB patients. We also found the prognostic efficacy of tumor miRNA level was consistent in Asians (HR: 0.53; 95% CI: 0.26-1.08; p = 0.0026) and non -Asians patients with MB (HR: 0.92; 95% CI: 0.26-3.22; p = 0.953). Results show that no significant publication bias was found in the analysis. Sensitivity analysis shows that our conclusions are robust and reliable. The included studies were assessed as good quality using quality evaluation methods. Shaw p et al. [37] the study suggests that increased overall pooled effect of miRNA expression is associated with poor overall survival in nasopharyngeal carcinoma (NPC) patients. in our study. However, some miRNAs expression was up-regulated in MB patients, and some were down-regulated. But in conclusion, the overall pooled effect of miRNAs expression in MB may predict better survival. Based on the limited original data available, the role of miRNAs