MiR-24-3p as a prognostic indicator for multiple cancers: from a meta-analysis view

Abstract A growing number of researches suggest that microRNAs (miRNAs) as oncogene or tumor suppressor genes play a fundamental role in various kinds of cancers. Among them, miR-24-3p, as a star molecule, is widely studied. However, the prognostic value of miR-24-3p is unclear and controversial. We conducted this meta-analysis to evaluate the prognostic value of miR-24-3p in a variety of cancers by integrated existing articles from four databases. PubMed, Embase, Web of Science, and Cochrane Library (last update in March 2020) were searched for approach literature. Hazard ratios (HRs) and odds ratios (ORs) were used to evaluate the association between miR-24-3p expression levels and prognostic value or clinicopathological characteristics, respectively. A total of 15 studies from 14 literature were finally qualified and concluded in the present meta-analysis. A significantly worse overall survival was observed in higher expression of miR-24-3p cancer group for OS (overall survival) of log-rank tests and Cox multivariate regression by fixed effects model. Also, we found a significant correlation between elevated miR-24-3p levels to RFS (recurrence-free survival) and DFS (disease-free survival). In addition, the pooled odds ratios (ORs) showed that evaluated miR-24-3p was also associated with the larger tumor size (≥5 cm) and advanced TNM stage (III and IV). Built on the above findings, elevated expression levels of miR-24-3p may serve as a promising biomarker used to predict the worse prognosis of cancer patients.

MiR-24-3p (used name was miR-24), a master regulator from the gene cluster of miR-23a-24-27a, has been identified as an onco or oncosuppressor-miR and its expression is closely associated with cancer occurrence and development by recent studies [24,25]. Previous studies showed that miR-24-3p was highly expressed in many carcinomas [26,27]. In addition, the evaluated expression of miR-24-3p was also found to be associated with cancer prognosis and tumor clinicopathological features, but there were some opposite consequences [28,29]. Up to now, a number of studies have been investigated this molecule in many kinds of cancer, but the most individual study has their limits, for example, small sample size or obtaining controversial results, and so on.
Accordingly, to explore the clinical prognostic value of miR-24-3p in various cancers, we performed this systematic review and meta-analysis to give a better understanding.
To explore the source of the heterogeneity, the sensitivity analysis was performed, but there was also no positive consequence ( Figure 2C). Subsequently, funnel plots, Begg's test and Egger's test were implemented to assess the potential publication bias and two studies as the outliers were identified eventually ( Figure 2D; Liu et al. [41] and Gao et al. [29]). After removing them, dramatically decline of the heterogeneity was observed (I 2 = 34.60%, P=0.141) in the overall analysis, and the significance of the prognostic effects of miR-24-3p expression was still obvious ( Figure 2B). Finally, to further explore the source of the heterogeneity, subgroup analyses were applied by factors including population (Asian(Chinese) and Non-Asian), sample size (≥100 and <100), NOS scores (≥8 and     <8), specimen (tissue and non-tissue) tumor category 1 (solid tumor and non-solid tumor), tumor category 2 (digestive system and non-digestive system), and tumor (esophageal cancer, osteosarcoma, lung cancer, gastric cancer, colorectal cancer, ALL, AML, and hepatocellular carcinoma) ( Table 2). As a consequence, the heterogeneity was controlled successfully in six subgroups and all them have significant correlations: (1) The subgroup of non-Asian (HR = 2.615, CI: 1.668-4.099; I 2 = 0.000%, P=0. 693, Figure 3A). (2) The specimen derived from non-tissue (HR = 2.399, CI: 1.659-3.470; I 2 = 0.000%, P=0.949, Figure 3D). (3) The sample size greater than or equal to 100 (HR = 2.779, CI:2.051-3.766; I 2 = 0.000%, P=0 .873, Figure 3B). In addition, significant correlations are also observed in the study of NOS score less than eight by random effects model, which were consistent with the significance of the results by fixed effects model (Table 2). Moreover, significant correlations were observed between miR-24-3p expression levels and OS in the studies with the population derived from Asian (Chinese) (HR = 1.381, CI: 1.219-2.004, Figure 3A), solid tumor (HR = 1.448, CI: 1.131-1.852, Figure 3E), digestive system (HR = 1.705, CI: 1.291-2.253, Figure 3F) and non-digestive system (HR = 1.461, CI: 1.021-2.090, Figure 3F) by fixed effects model, while there were no significances identified in these groups when the random effects model was applied ( . Due to insufficient data, the consequence was lack of efficiency and the heterogeneity was also significant (I 2 = 93.00%, P≤0.001). Therefore, more relevant studies are required to perform the analysis. Built on the above consequences, meta-regression was also used, but there was no meaningful contribution identified with impacting on the heterogeneity (Table 2).

The independent role of miR-24-3p expression levels as a prognostic indicator
Five studies containing 775 patients implemented the Cox multivariate regression to assess the prognostic value of miR-24-3p expression levels in carcinoma patients by adjusting other factors. The significant correlation of miR-24-3p expression levels to the OS (HR = 2.384, CI: 1.813-3.134) was observed by fixed effects model. However, the heterogeneity was relatively obvious (I 2 = 82.30%, P=0.000,  (Table 3). But the sensitivity analysis suggested that Gao et al. [29] has a significant impact on the result ( Figure 4C). The heterogeneity was vanishing (I 2 = 0.000%, P=0.591, Figure 4B) by removing this outlier and the correlation of miR-24-3p expression levels to the OS was also significant (HR = 3.039, CI: 2.268-4.074, Figure 4B). Finally, funnel plots, Begg's test (P=0.734) and Egger's test (P=0.460) indicated that there was no bias. But, the number of enrolled studies was few and more data are needed to reinforce this result.

The correlation of miR-24-3p expression levels to the RFS /DFS
Except for OS as a prognostic indicator, RFS and DFS are also be accepted as an evaluation criterion. Here, four studies reported RFS including 393 patients applied log-rank tests, while only one also utilized Cox multivariate regression. After pooling the HR, we observed a significant association between miR-24-3p expression levels to the RFS of log-rank tests (HR = 2.315, CI: 1.491-3.594, Figure 5A) by fixed effects model. However, the heterogeneities were quite obvious (I 2 = 66.70%, P=0.290, Table 4). The random effects model was further implemented but the     , indicating that the heterogeneity influenced the consequences significantly. Furthermore, owing to the limited number of statistics from Cox multivariate regression, the sensitivity analysis and publication bias were only applied to analysis with data extracted from log-rank tests. The sensitivity analysis result indicated that no studies had significant influences on the consequent ( Figure 5C). However, the investigation of potential publication bias identified an outlier ( Figure 5D, Wang S. et al. [40]). After deleting this study, the heterogeneity was obvious declined (I 2 = 45. 30%, P=0.161) and the significance of the correlation between miR-24-3p expression levels and the RFS was not altered (HR = 2.575, CI: 1.642-4.029, Figure 5B). Due to the limit included studies, more data are needed to enhance the result. In addition, there were only two studies containing 226 patients revealed the DFS statistics and almost no heterogeneity in both log-rank tests and Cox multivariate regression (I 2 = 3.600%, P=0.309, I 2 =0.000%, P=0.330, respectively, Table 4) by used a fixed effects model. We also

Correlations between miR-24-3p levels and clinicopathological features among various carcinomas
Six studies containing 536 patients investigated the correlation of miR-24-3p expression levels to different clinical characteristics. As showed in Table 5 .50%, P=0.000, respectively). To decrease the heterogeneity, sensitivity analysis and publication bias were further investigated to each of them. As a result, an outlier was found (Liu et al. [36]) in the TNM stage. After removing the outlier, the heterogeneity was dramatically decreased from 85.50% to 0.000% and the associations between high miR-24-3p expression levels to advanced TNM stage were significant (OR = 2.328, CI: 1.490-3.637) ( Figure 6). Moreover, there was no potential publication bias about TNM stage by funnel plot, Begg's test (P=0.086) and Egger's test (P=0.734). For the analyze of lymph node metastasis, there were only two studies and had an obvious opposite result (Pan et al. [33], OR = 2.974, CI: 1.101-8.037; Zhou et al. [35], OR = 0.725, CI: 0.238-2.208). Due to insufficient data, the consequence would be lack of efficiency and the reasons of heterogeneity were unacceptable. Thus, more pertinent studies are required to perform the analysis.

Discussion
It is of great importance to explore prognostic biomarkers with the patients of carcinoma as specific biomarkers can further help to directly stratify patients and effectively guide clinical decision-making. MiR-24-3p, as an oncogene or tumor suppressor, plays a key role in the occurrence, progression and metastasis of human carcinoma was realized by more and more researchers gradually [24,25]. Quan et al. [45] had made a meta-analysis to research the correlation between miR-23a/24-2/27a cluster with human cancers, but they only had limited data to draw a conclusion that high expression levels of miR-23a/24-2/27a indicated a worse prognosis and no further analyzing the correlation between miR-24-3p expression levels to the clinicopathological characteristics. Subsequently, more and more studies that focus on the miR-24-3p expression levels with cancer progression, metastasis and prognosis of patients were carried on [4][5][6][7][14][15][16]. Thus, the exact role of miR-24-3p on the clinical prognosis of patients in various human carcinomas still need to investigate further. In this meta-analysis, total of 15 studies including 1518 people were recruited. Among them, ten studies containing 1212 patients provided the statistics of the OS by log-rank tests. By the pooling strategy, we know that the elevated miR-24-3p expression levels were linked to worse prognosis of cancer patients. Subsequently, several approaches were put in place to investigate the heterogeneity. First, subgroup analyses were conducted to identify the potential sources of heterogeneity. We found that the heterogeneity was achieved within the non-Asian population, non-tissue, the studies of sample size greater than or equal 100, hematologic tumor, hepatocellular carcinoma and lung cancer. But it was not controlled in other groups, such as the Asian population, studies   of sample size less than to 100, solid tumor and so on. Second, the sensitivity analysis was used, but no outlier was identified to impact on the pooled results significantly. Third, two studies were identified as outliers by publication bias evaluation (Liu et al. [41] and Gao et al. [29]). After retrieving the data of outliers, we found that the specimen recruited in them were all from Asian (Chinese), tissue and the sample size less than 100 which all have strong heterogeneity. Besides, Liu et al. [41] was the sole study that focused on Osteosarcoma and Gao et al. [29] had an opposite conclusion with Kerimis et al. [38] who also investigated the miR-24-3p expression levels to colorectal cancer. After removing those two outliers, greatly declined of the heterogeneity was observed. Based on the mentioned above, these two studies could be the major sources of heterogeneity. However, more relevant data are needed to further investigate because of the limit number of studies. There were five studies including 775 patients obtained the data of HRs by Cox multivariate regression. Cox multivariate regression has been known as an effective approach because it can evaluate the contribution of each factor independently by adjusting other factors [46]. Thus, the consequences by Cox multivariate regression are always considered as independent effects of each factor on the clinical outcome. As a result, we found that the significance was inconsistent among different effects model. This phenomenon suggested that the heterogeneity was relatively obvious and the consequences were instable. Through the subgroup analyses, we found that the heterogeneity was declined in hepatocellular carcinoma and achieved in the studies of sample size larger than or equal 100. In addition, the sensitivity analysis identified one outlier, Gao et al. [29] who has an opposite conclusion with Kerimis et al. [38]. After removing this study, the heterogeneity had been significantly vanished.
High miR-24-3p expression had a significantly worse survival and there was no publication bias. Thus, the power of miR-24-3p expression levels might serve as an independent prognostic indicator and we need more data to reinforce this conclusion. Also, we detected additional indexes, such as RFS and DFS. MiR-24-3p expression levels were deemed to be significantly associated with DFS of statistics extracted from both log-rank tests and Cox regression analysis. For the RFS of cancer patients, only the fixed effects model revealed a significant correlation between miR-24-3p expression with this prognostic index and the heterogeneity was palpable. We identified an outlier (Wang et al. [40]) through publication bias evaluation. After removing the present study, the heterogeneity was declined and the significance of the association between miR-24-3p expression levels and the RFS was not altered. As for the clinicopathological parameters, six studies including 536 patients had evaluated the association of miR-24-3p expression levels to the distinctive clinical parameters. The overexpression of miR-24-3p was found to be significantly related to larger tumor size by the fixed effects model. Moreover, we found significant heterogeneity between miR-24-3p expression levels to TNM stage. Appling sensitivity analyses, we identified one study (Liu et al. [36]) that had great impact on the result for the TNM stage. After removing this study, the heterogeneity completely disappeared; the association between miR-24-3p expression levels to the TNM stage was also significant. In addition, there were only two studies about the lymph node metastasis are enrolled and the conclusion might be not reliable. The analyzes of clinical features of a definite carcinoma should be normalized for the cut-off values, the feature categories and so on, to enrich the enrolled cases and characteristics for the meta-analysis.
As far as we know, this meta-analysis was the most comprehensive and systematic one to explore the correlation between the miR-24-3p expression levels with the prognosis of cancer patients in depth. Subgroup analysis, meta regression, sensitivity analysis and publication bias had been used to investigate the possible source of the heterogeneity to the greatest extent [47]. In spite of this, several flaws were hard to avoid in this meta-analysis. First, inevitable limitation from insufficient data in this analysis (only 15 studies with 1518 patients). Second, the cut-off values of the miR-24-3p expression levels were not exactly among those studies, thus, the accuracy of prognostic results may be influenced. Third, part of HRs was calculated from the survival curves which may cause some bias. Four, the number of recruited studies for DFS, RFS and clinicopathological features analyses was relatively insufficient. Taking above reasons into account, we need better designed and large sample size studies for further research before applying miR-24-3p as a prognostic biomarker of tumor in clinical applications.

Conclusions
The overexpression of miR-24-3p was an underlying risk of poor prognosis in various human carcinomas, especially in hepatocellular carcinoma and lung cancer. As for other types of carcinomas, the results are not yet stable and more studies including normalized research conditions are required to identify miR-24-3p prognostic values further. In addition, high miR-24-3p expression levels were linked to the progression of cancers, developing more malignant behaviour, such as larger tumor sizes and the advanced TNM stages. To sum up, miR-24-3p expression levels could serve as a potential prognostic marker of human carcinoma.