Figures & data
Table 1. Sequences of shRNAs
Figure 1. LINC00857 promotes malignant phenotypes of PAAD cells.
(a) The expression level of LINC00857 expressions were detected through RT-qPCR in HPDE and three types of PAAD cells (PANC-1, Capan-1, and BxPC-3). (b) The efficiency of LINC00857 knockdown in PANC-1 and BxPC-3 cells was tested by RT-qPCR. (c) Wound healing assays were conducted in PANC-1 and BxPC-3 cells to understand the influence of LINC00857 on cell migration capabilities. N = 3. One-way ANOVA followed by Dunnett’s test. (d-e) Transwell assays were performed in PANC-1 and BxPC-3 cells to test the cell migration and invasion capabilities before and after LINC00857 knockdown. (f) The spindle shaped cells were reduced after LINC00857 knockdown. (g) The expressions of key EMT-related proteins were measured via western blot in PANC-1 and BxPC-3 cells when LINC00857 was knocked down. (h) FISH was performed to locate LINC00857 in PANC-1 and BxPC-3 cells. *P < 0.05, **P < 0.01.
Figure 2. LINC00857 regulates the expression of CLDN12 by sponging miR-150-5p in PAAD cells.
(a) RIP assays were performed in PANC-1 and BxPC-3 cells and the enrichment of LINC00857 was quantified by RT-qPCR. (b) The potential candidate miRNAs were predicted on LncBase Predicted v.2 website. (c) RNA pull-down assays were done in PANC-1 cells and the enrichments of three candidate miRNAs were measured by RT-qPCR. (d) After RNA pull-down assays, RT-qPCR was performed in PANC-1 cells to detect the enrichment of miR-150-5p under different conditions. (e) RIP assays were performed in PANC-1 cells and the enrichments of miR-150-5p as well as LINC00857 were quantified via RT-qPCR. (f) Four candidate mRNAs likely to interact with miR-150-5p were screened out from starBase v2.0 website. (g) The expressions of four candidate mRNAs were measured in PANC-1 cells before and after LINC00857 knockdown by RT-qPCR. (h) RNA pull-down assays and RT-qPCR were implemented in PANC-1 cells to quantify the enrichment of miR-150-5p under different conditions. (I) RIP assays were performed and the enrichments of miR-150-5p and CLDN12 were quantified in PANC-1 cells through RT-qPCR. (j) Before and after LINC00857 knockdown, the expressions of CLDN12 and miR-150-5p were respectively detected in PANC-1 cells by RT-qPCR. (k) Luciferase reporter assays were performed in PANC-1 cells to confirm if miR-150-5p could bind to the wild-type of LINC00857 and CLDN12-3ʹUTR. (l-m) Rescue experiments were conducted in PANC-1 cells and the expression and protein levels of CLDN12 were measured through RT-qPCR and western blot. **P < 0.01.
Figure 3. CLDN12 facilitates the malignant phenotypes of PAAD cells.
(a) The efficiency of sh1/2/3-CLDN12 was tested in PANC-1 cells by RT-qPCR. (b-c) Wound healing and Transwell assays were performed in PANC-1 cells to evaluate the impacts of CLDN12 silence on cell migration and invasion abilities. (d) The changes of PANC-1 cell shape were observed before and after CLDN12 silence. (e) The expressions of key EMT-related proteins were examined in PANC-1 cells through western blot. **P < 0.01.
Figure 4. LINC00857 recruits SRSF1 for alternative splicing to regulate CLDN12 expression in PAAD cells.
(a) Potential RBPs likely to bind to LINC00857 and CLDN12 were predicted by searching on starBase v2.0 website. (b) RNA pull-down and western blot assays were performed in PANC-1 cells to verify the combination between relevant RNAs and predicted RBPs. (c) After RIP assays, the enrichments of LINC00857, CLDN12 and pre-CLDN12 were quantified in Anti-SRSF1 group by RT-qPCR. (d) By searching on NCBI website, three transcript variants of CLDN12 and one translated protein were found out. (e) The efficiency of sh-SRSF1 was examined in PANC-1 cells by RT-qPCR. (f) CLDN12 expression was quantified via RT-qPCR before and after SRSF1 silence in PANC-1 cells. (g) The expressions of three transcript variants were detected by AGE and RT-qPCR before and after SRSF1 silence in PANC-1 cells. (h) The protein level of CLDN12 was examined through western blot when SRSF1 was silenced in PANC-1 cells. (i-j) Flag-CLDN12a-GST, Myc-CLDN12c-GST, Myc-CLDN12c-GST were co-transfected into 293 T cells; RT-qPCR and western blot were applied to detect the RNA and protein levels of CLDN12 in the transfected cells separately. (k) The binding sites between SRSF1 and CLDN12 were predicted via ESEfinder and the mutant binding sites were designed as Mut1 and Mut2. (l) By AGE and RT-qPCR, the mRNA levels of three transcript variants of CLDN12 were tested under different conditions in PANC-1 cells. (m) The expression of three transcript variants of CLDN12 was measured via AGE and RT-qPCR before and after the knockdown of LINC00857. (n) The expression of SRSF1 was detected in the transfected PANC-1 cells with sh1-LINC00857 or sh2-LINC00857. (o) RNA pull-down assays were conducted in PANC-1 cells and the protein level of SRSF1 was measured before and after LINC00857 knockdown through western blot. (p) RT-qPCR was applied to verify the overexpression efficiency of SRSF1. (q) The mRNA levels of three transcript variants of CLDN12 were assessed under different conditions in PANC-1 cells. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5. ZNF460 as the transcription factor of LINC00857 promoter regulates LINC00857 expression in PAAD cells.
(a) Potential transcription factors of LINC00857 promoter were projected through linking UCSC database with JASPAR website. (b) DNA pull-down assays were implemented in PANC-1 cells and western blot analysis helped determine the possible transcription factor of LINC00857 promoter. (c) The overexpression efficiency of pcDNA3.1-ZNF460 was verified through RT-qPCR. (d) Luciferase reporter assays were performed to confirm the binding relationship between ZNF460 and LINC00857 promoter in PANC-1 cells. (e) The efficiency of ZNF460 knockdown was examined in PANC-1 cells by RT-qPCR. (f) LINC00857 expression was detected before and after ZNF460 downregulation in PANC-1 cells through RT-qPCR. (g) The shapes of PANC-1 cells were observed to determine the effects of ZNF460 downregulation on EMT of PAAD cells. (h) The expressions of key proteins associated with EMT were measured through western blot analysis. **P < 0.01, ***P < 0.001.
Supplemental material