3.1 FAM83A-AS1 is up-regulated in ESCC tissues
The bioinformatics data analysis retrieved from GEPIA (http://gepia2.cancer-pku.cn/#index) and lnCAR[16] revealed that FAM83A-AS1 was remarkably increased in ESCC (Fig. 1A-B). Our results also corroborated in the high expression of FAM83A-AS1 in 51 paired ESCC tissues compared with adjacent non-tumor tissues (Fig. 1C). Next, we collected the clinical data of these independent ESCC cases and assessed the association between FAM83A-AS1 level and clinical parameters of ESCC patients. Here, we identified FAM83A-AS1 expression was significantly correlated with differentiation grade (P = 0.0209 and advanced stages (P=0.0104, Table 1)). However, there was no obvious association in other clinical parameters, including gender, age, and so on. Additionally, we assessed the diagnostic and prognostic effects of FAM83A-AS1 in ESCC. Kaplan-Meier statistics suggested that patients with high expression of FAM83A-AS1 exhibited a similar overall survival time with those with low FAM83A-AS1 levels (Fig. 1E). Whereas ROC analysis showed that FAM83A-AS1 gained high accuracy in distinguishing ESCC (Fig. 1D). The AUC(Area under the curve) was 0.954 (95% CI 0.895–0.985) and sensitivity and specificity were 92.59% and 90.57%, respectively. Taken together, these findings implied that FAM83A-AS1 plays a tumorigenic role in ESCC development and progression.
3.2 FAM83A-AS1 accelerated ESCC cell growth.
To seek for the precise function of FAM83A-AS1 in ESCC cells, we designed three siRNA targeting FAM83A-AS1 and a negative control sequence and named them as si-FAM83A-AS1#1, si-FAM83A-AS1#2, si-FAM83A-AS1#3, and si-NC. Next, we assessed the gene silencing efficiency by qRT-PCR assays in KYSE30 and EC109 cells. Clearly, si-FAM83A-AS1#2 and si-FAM83A-AS#3 exhibited higher gene silencing efficiency (Fig. 2A). Consequently, we focused on these two siRNAs for the following experiments. CCK-8 results demonstrated that cell proliferation ability was dramatically blunted after depleting FAM83A-AS1 in KYSE30 and EC109 cells (Fig. 2B-C). These data suggested that FAM83A-AS1regulated cell proliferation of ESCC cells in vitro.
3.3 FAM83A-AS1 enhances ESCC cells metastasis capabilities
Given the importance of FAM83A-AS1 in ESCC, we assessed the effect of FAM83A-AS1 on ESCC cell migration and invasion. Transwell migration and invasion assays were performed according to the description before [17]. Interestingly, we observed that KYSE30 cell migration capacity was significantly attenuated following knocking down FAM83A-AS1. Similarly, the migration assays in EC109 cells produced the same results (3A-C). Furthermore, the Transwell invasion assay indicated that the invasive capabilities of both KYSE30 and EC109 cells exhibited a decreasing trend in si-FAM83A-AS1 compared to the si-NC group, which was consistent with the migration assays. Overall, these investigations revealed that FAM83A-AS1 promoted ESCC metastasis process.
3.4 FAM83A-AS1 amplifies ESCC cells apoptosis events
Annexin V/PI double staining assay was adopted to evaluate the influence FAM83A-AS1 on ESCC cells apoptosis progress. Correspondingly, we examined apoptosis events in ESCC cells using flow cell cytometry. Notably, as shown in Fig. 4A-D, we discovered that the percentage of cells in KYSE30 cells had an increase in KYSE30 cells after FAM83A-AS1 depletion. Meanwhile, inhibiting FAM83A-AS1 also led to the growth of the proportion of apoptosis cells in EC109 cells. (Fig. 4E-H). Beyond these findings, we argued that FAM83A-AS1 regulated ESCC cell apoptosis.
3.5 FAM83A-AS1 expediates ESCC cells cycle distribution
To explore the potential mechanism of FAM83A-AS1 in promoting ESCC cell proliferation, we performed cell cycle assays based on PI staining with flow cell cytometry. The analysis results illustrated that FAM83A-AS1 remarkably regulated cell cycle distribution. Specifically, the population of G1 phase cells was strikingly augmented in the FAM83A-AS1-deficient group compared with the control group. In contrast, the cells in the S phase were sharply reduced after the silencing of FAM83A-AS1 (Fig. 5A-D). Taken together, these observations suggested that FAM83A-AS1 promoted cell proliferation through accelerating cell cycle process.
3.6 FAM83A-AS1 regulates miR-214 expression pattern in ESCC cells
It was conclusively established that lncRNA could act as competing endogenous RNA by binding miRNAs, leading to the liberation of target mRNAs [18,19]. First, we analyzed the location of FAM83A-AS1 in cells. Results indicated that FAM83A-AS1 was mainly accumulated in the cytoplasm (Fig. 6A). Moreover, we predicted potential miRNA candidates of FAM83A-AS1 by the Annolnc website. Here we found that miR-214 was a target miRNA of FAM83A-AS1. Additionally, it was reported that miR-214 suppressed ESCC progression by regulating cell proliferation, migration, and invasion [20]. Therefore, we speculated that FAM83A-AS1 could regulate miR-214 expression to promote ESCC progression. As illustrated in 6B, there was an impressive increase of miR-214 expression level after FAM83A-AS1 depletion in KYSE30 and EC109 cells. Subsequently, miR-214 expression was negativity correlated with FAM83A-AS1 in ESCC tissues (6C, R2=0.6836, P<0.0001). To confirm the role of miR-214 in ESCC cells, we designed the miR-214 inhibitor and the corresponding control sequence. As shown in 6D, miR-214 expression was decreased after transfected with miR-214 inhibitor compared with the control group. Interestingly, we noted that cell migration potential was increased after FAM83A-AS1 knockdown combined with miR-214 inhibition compared with FAM83A-AS1 depletion (Fig. 6E-F). Collectively, these results illuminated that miR-214 could partially rescue the inhibition of cell migration caused by FAM83A-AS1 silencing. Overall FAM83A-AS1 drives ESCC progression by regulating miR-214.
3.7 The role of CDC25B in ESCC and the association between CDC25B, miR-214, and FAM83A-AS1
CDC25B was reported to be the direct target of miR-214 [21]. CDC25B was dramatically elevated in multiple cancer, which was validated by the pan-cancer analysis from GEDS [22] and Uclcan [23] (Fig. 7A,7C). Notably, the CDC25B expression level was significantly higher in ESCC tissues. In addition, the data retrieved from GEPIA, Uclcan, and oncomine supported that CDC25B was predominantly accumulated in ESCC tissues compared to normal esophagus tissues (Fig. 7B, 7D, 7E). These investigations provided evidence that CDC25B was an important driving force in ESCC development. Moreover, we found that CDC25B obtained a high diagnostic accuracy for ESCC (Fig. 7F, AUC=0.9263, P<0.0001). Interestingly, the CDC25B expression pattern was increased after inhibiting miR-214 in ESCC cells (Fig. 7G). In contrast, the CDC25B level exhibited a decreasing trend when ESCC cells were treated was si-FAM83A-AS1. In summary, FAM83A-AS1 up-regulated CDC25B expression by regulating miR-214 as molecular sponge.