Meta-analysis and trial sequential analysis of LncRNA H19 polymorphic variants on susceptibility to cancer

Background: Although myriad researches upon the associations between LncRNA H19 polymorphic variants (rs2839698 and the susceptibility to cancer have been conducted, these results remained contradictory and perplexing. Basing on that, a systematic review and updated meta-analysis was conducted to anticipate a fairly precise assessment about these associations. Methods: We retrieved the electronic databases EMBASE, PubMed and Web of Science for valuable academic studies before October 1st, 2019. Ultimately, 24 of which were encompassed after screening, and the available data was extracted and integrated. The pooled odds ratios (ORs) with 95% confidence intervals (CIs) was adopted to evaluate the strength of these associations. For multi-level investigation, subgroup analysis derived from source of controls together with genotypic method was preformed. Eventually, 24 articles altogether embodying 52 studies were included. Results: The results illuminated that LncRNA H19 SNPs mentioned above were all irrelevant to cancer susceptibility. Nevertheless, crucial results were found concentrated in population-based control group when subgroup analysis by source of controls were performed in H19 mutation rs2839698 and rs2735971. Meanwhile, in the stratification analysis by genotypic method, apparent cancer risks were discovered by TaqMan method in H19 mutation rs2107425 and rs3024270. Then, trial sequential analysis (TSA) demonstrated that the results about such associations were firm evidence of effect, except rs2735971 polymorphism. Conclusion: Therefore, this meta-analysis indicated that LncRNA H19 SNPs were not associated with the susceptibility to human cancer. However, after the stratification analysis, inconsistent results still existed in different genotypic method and source of control. Thus, more high-quality studies on cancer patients of different factors were needed to confirm these findings.

evaluate the gene and genotype frequency, and P value exceeded 0.05, guaranteeing a significant equilibrium. In addition, considering the examination of the stability and reliability of the results, sensitive analysis was adopted through consecutively omitting one study per time. Meanwhile, we conducted both Egger's test and Begg's funnel plots in order to verify the publication bias among these studies Statistical data was analyzed through Stata statistical software (Version 12.0, Stata Corporation, College Station, TX, USA).

Trial sequential analysis
The results of the meta-analysis should relate the total number of randomized participants accounting for statistical diversity to avoid type I errors. Thus, TSA was performed to estimate the required information size. TSA could confirm greater statistical data reliability through modifying the threshold with dispersive data for significance level. We then calculated the required information size and constructed the trial sequential monitoring boundaries (the sloping red line). If the blue line (representing the cumulative Z-curve) crossed the sloping red line or exceeded the vertical red line (representing the information size required), a sufficient information size would be guaranteed and crucial result would be reached. On the contrary, either of the red line not being reached reveals that additional clinical trials were necessary to guarantee the sufficient information size for further verification. The TSA software (TSA, version 0.9, 2011; Copenhagen Trial Unit, Copenhagen, Denmark) was adopted in this study.

Studies characteristics
Primitively, a total of 182 articles were collected under the guidance of the retrieve strategy above for further screening. Then, 24 articles containing 52 studies met the inclusive criteria, ranging from  Table 1. Asians, Caucasians, Africans and Mixed were involved in these studies. We separated these studies into population-based (PB) and hospitalbased (HB) groups to help differentiate between various sources of control. Moreover, the studies collected performed eight genotypic methods altogether, for instance, Illumina, Taqman, Sequenom, etc.
Meanwhile, we calculated the pooled ORs and 95% CIs using five genetic model in order to assess the association between lncRNA H19 ploymorphisms and cancer susceptibility, results of which were tabulated in Table 2. Also, stratification analysis by source of controls along with genotypic method was applied to investigate the heterogeneity of all studies.

rs2839698 GA and cancer susceptibility
Thirteen studies about lncRNA H19 rs2839698 G A ploymorphism and the susceptibility to cancer consisting 7,351 cases and 10,066 controls met the inclusive criteria. The pooled ORs were 1.08 (95%  (Figure 3). Besides, no positive results were observed in either of the subgroup analysis by source of control and genotypic method.

rs2107425 CT and cancer susceptibility
A total of 11 studies embodying 17,628 controls and 12,292 cases were investigated to LncRNA H19 polymorphic variants rs2107425 C T and the susceptibility to cancer. Analogously, no significant association between rs2107425 C T polymorphism and cancer risk was shown in the meta-analysis  (Figure 4). Nevertheless, as to the stratification analysis of genotypic method, the result was significant only in TaqMan (allele model: OR=0.86, 95% CI=0.80-0.94), while no significant results was detected in subgroup of source of control.

rs2735971 AG and cancer susceptibility
The present meta-analysis enrolled 4,393 controls and 3,522 cases from a sum of six studies on

Sensitivity analysis
We performed sensitivity analysis by excluding single eligible study sequentially to detect individual study's influence on the pooled results. According to the results, no single study was found affect the pooled OR in the allele model, suggesting a statistically robust results (Figure 7).

Publication bias
Both Egger's test and Begg's funnel plot were utilized in the selected literature. With the symmetrical shapes of funnel plots shown in Figure 8

Trial sequential analysis results
In this meta-analysis, Figure 9 showed that the information sizes of all the H19 mutations investigated were sufficient except rs2735971 A G polymorphism, indicating that the results about the associations between LncRNA H19 polymorphic variants (rs2839698 G A, rs3024270 C G, rs2107425 C T and rs217727 G A) and the susceptibility to cancer were firm evidence of effect, and the further verification is necessary for the relationship of rs2735971 A G mutation and cancer risk. . The various cancer location and patient ethnicity probably account for the discrepancies amongst these studies. In these studies, a small sample size and controversial results caused by the former factor might make these analysis relatively unreliable. Herein, we conducted this metaanalysis with the largest sample capacity and the most up-to-date studies and data, comprehensively analyzing all literatures to study such association. According to quantitative synthesis results, all the mutation mentioned above were found no significant association.

Discussion
While stratified by source of control, significant association was found in the PB control group between rs2839698, rs2735971 and rs3024270 polymorphisms and the susceptibility to cancer, whereas no significant results were detected in their HB control group. Lack of the representativeness might account for the phenomena. Moreover, referring to subgroup analysis of genotypic method, significant association was also detected between the risk of cancer and rs2107425, rs3024270 polymorphisms adopting TaqMan method for genotyping, whereas similar results were not found while using other genotypic methods. The possible reason might be that different genotypic methods lead to different statistical results owing to their relative merits. The merits of TaqMan are the lower probability of PCR pollution due to that the reaction happens in PCR process, avoiding separation and elution [45].
TSA, as an statistical tool, is similar to interim analysis in a single trial, where trial monitoring boundaries are drawn for each outcome whether to continue additional trials to assess for evidence while a P value is small enough to show the projected effect or for futility [46]. The association shown in the results of this meta-analysis could be unreliable accounting for limited data. Therefore, TSA was adopted in order to diminish the probability of type I error and verify whether the evidence of our results was adequate or not. The results about the associations between LncRNA H19 polymorphic variants (rs2839698, rs217727, rs2107425 and rs3024270) and the susceptibility to cancer were firm evidence of effect. However, the cumulative Z-curve of H19 rs2735971 polymorphism neither crossed the trial sequential monitoring boundary nor exceeded the required information size, indicating that false positive results might be eliminated in advance due to repeated significance testing and random errors in meta-analysis [47]. In order to obtain results with firm evidence, further verification is necessary.
Inevitably, several additional limitations should be warranted in this meta-analysis. (1). As a multifactorial disease, overall cancers are influenced by genetic combined with environmental factors.
Focusing on single gene region, this meta-analysis ignored the complex interaction between various factors, in which case the association was unilateral; (2). The scale of studies collected was fairly narrow, referring to the subgroup analysis. Also, the amount of these studies needs to be expanded. Subgroups with less than three studies were retained, thus might causing the potential false associations; (3). The ethnicities of studies were concentrated on Asian , which means the results might not applicable for all population. Hence, to ensure reliability of our meta-analysis, attention must be paid to the influence of various factors in the future studies of more large-sample, multicenter and high-quality researches.

Conclusion
To conclude, the results of this meta-analysis revealed that five H19 SNPs (rs2839698 G A, rs3024270 C G, rs2107425 C T, rs2735971 A G and rs217727 G A) had no significant association with the overall cancer susceptibility, thereby suggesting that H19 might not have clinical significance in cancer diagnosis and treatment. However, after the stratification analysis, inconsistent results still existed in different genotypic method and source of control. Thus, more high-quality studies on cancer patients of different factors were needed to confirm these findings.

Ethics approval and consent to participate
None declared.

Consent for publication
Written informed consent for publication was obtained from all participants.

Availability of data and material
All data on which the conclusions rely are either deposited in publicly available repositories or presented in the main papers or additional supporting files.

Competing interests
We declare that we have no conflict of interest.   Figure 1 The flow diagram of literature retrieval and screening process.        TSA of the association between H19 polymorphisms and the susceptibility to cancer. The calculation of the required information size was based on a 2-sided α= 5%, β= 15% (power 85%), and a relative risk reduction of 20%. A: rs2839698; B: rs217727; C: rs2107425; D: