Down regulation of multidrug resistance-associated protein 4 expression by RNA interference enhances radiosensitivity of colorectal carcinoma cell lines in vitro / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the effect of multidrug resistance-associated protein 4 (MRP4) expression on the radiosensitivity of colorectal carcinoma cell lines in vitro.</p><p><b>METHODS</b>The vector of shRNA for RNA interference was constructed and then transfected into HCT116 cell line to steadily down-regulate the expression of MRP4. HCT116 cells were divided into 3 groups including the CON group(non-transfected), NC group (negative control virus was added), and KD group (RNAi target was added for transfection). To test the effectiveness of RNA interference, real-time polymerase chain reaction and Western blot were used to measure the expression pattern of MRP4 at both mRNA and protein levels, respectively. For the examination of the effect of RNA interference of MRP4 on the radiosensitivity, flow cytometry was used to calculate the rate of apoptotic cells 24 h after 4 Gy radiation. Proliferation of the cells was measured via MTT assay at different time points.</p><p><b>RESULTS</b>ShRNA plasmid was successfully constructed. Transfection of this constructed vector into HCT116 cell line caused steady silencing of MRP4 expression (HCT116-KD). MRP4 mRNA and protein expression were significantly down-regulated following RNA interference(P<0.05). Twenty-four hours after radiation, the apoptosis rate of KD cell line was (71.7±0.8)%, significantly higher than that in the CON group [(56.1±0.9)%] and NC group[(59.8±0.8)%](P<0.05). Fourty-eight hours and 72 hours after radiation, the proliferation was significantly inhibited in KD cells compared to the control groups(P<0.05).</p><p><b>CONCLUSIONS</b>Expression of MRP4 is closely related to radio-tolerance of colorectal carcinoma. Down-regulation of MRP4 expression by RNA interference enhances radiosensitivity of colorectal carcinoma cell lines in vitro. MRP4 may be an effective molecular marker for predicting the radiosensitivity of colorectal carcinoma.</p>

Long non-coding RNA influences radiosensitivity of colorectal carcinoma cell lines by regulating cyclin D1 expression / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To screen long non-coding RNA which influences radiosensitivity of colorectal carcinoma cell lines and investigate the mechanism.</p><p><b>METHODS</b>Under different doses of radiation, colony formation assay and single-hit multi-target model were conducted to draw dose-survival curve and SF2 value of colorectal carcinoma cell lines(RKO, Lovo) was calculated. High-throughput lncRNA/mRNA chips were used to screen lncRNA genes and protein coding genes with expression differences more than 2 folds between RKO, Lovo cell lines and RKO cell line receiving 2Gy radiation. The main action pathway was computed by Gene Ontology analysis combined with Pathway analysis in order to explore the mechanism which induces the effect of lncRNA on radiosensitivity of colorectal carcinoma cell lines. Further experiment on P53, P21, cyclin D1 expression contents of RKO cell line was confirmed by real-time RT-PCR.</p><p><b>RESULTS</b>Lovo(SF2=0.47) was more sensitivity to radiation than RKO(SF2=0.53) according to the outcome of colony formation assay. High-throughput lncRNA/mRNA chips identified a total of 268 lncRNA genes and 270 protein coding genes. Gene Ontology analysis showed that the expression of genes associated with cell cycle process were significantly different (38.6%). There was a significant relationship between expression of several lncRNAs and CCND1 gene. Real-time RT-PCR showed no significant differences of P53 and P21 expression in RKO and Lovo cell lines(P>0.05), while cyclin D1 expression of RKO cell line was higher than that of Lovo cell lines(P<0.05). After exposed to 2 Gy doses of radiation, there was an obvious decrease of cyclin D1 expression in RKO cell lines(P<0.05), while P53 and P21 expressions were not different(P>0.05).</p><p><b>CONCLUSION</b>The possible mechanism is that lncRNAs compose transcription compound to combine with CCND1 gene and influence radiosensitivity of colorectal carcinoma cell lines by regulating expression of cyclin D1, which is independent of P53-P21-cyclin D1 pathway.</p>

Competing endogenous RNA regulation mechanism and its role in the development and progression of colorectal cancer / 中华胃肠外科杂志

MicroRNAs are negative regulators of mRNA, and latest studies show that "mRNAs can also inhibit microRNAs". With these reciprocal interactions, different mRNAs with identical "microRNA binding site" cim regulate each other by competitively binding to the same microRNA pool. This is the novel competing endogenous RNA (ceRN A)regulating mechanism. The ceRN A mechanism, which is a totally new regulating mechanism , greatly expands the regulatory network across genes. It has been proved by experimental evidence that, in HCT116 colon cancer cells,KRAS and PTEN , ZEB2 and PTEN can regulate each other by ceRNA mechanism.