Role of the p53/miR-34a/SIRT1 Feedback Loop in Metformin-induced Radiosensitivity of Colorectal Cancer Cells.

Objective To study the effect of inhibiting the expression of FOXD1 gene on the radiosensitivity of colorectal cancer cells. Methods The expressions of FOXD1 mRNA and protein in human colorectal cancer tissues and cells were detected by Real-time PCR (qRT-PCR) and Western blot. The colorectal cancer cell line HCT116 was irradiated with 0, 2, 4 and 6 Gy of X-rays. The expression of FOXD1 in each groups were detected by qRT-PCR and Western blot. HCT116 cells were transfected with FOXD1 siRNA and its negative control and termed as si-FOXD1 group and si-NC group. When these cells were irradiated with 4 Gy X-rays, they were termed as si-FOXD1+ 4 Gy group and si-NC+ 4 Gy group. Cell proliferation was detected with MTT method, cell survival fraction was measured with colony formation assay, and DNA-PK activity was detected by TECT DNA-PK kit. The siRNA-transfected colorectal cancer cells were inoculated into BALB/c nude mice to establish the xenograft model. After irradiation, the volume and quality of the subcutaneous transplanted tumors were measured every 5 days. Results The expression of FOXD1 mRNA and protein in colorectal cancer tissues was higher than that in adjacent normal tissues (t=5.579, 4.816, P<0.05). The mRNA(t=5.85-17.62, P<0.05)and protein(t=9.04-11.42, P<0.05)expression of FOXD1 in different colorectal cancer cell lines was higher than that in colonic mucosa epithelial cell line NCM460. The expression of FOXD1 in colorectal cancer cells HCT116 was increased after radiation in a dose dependent manner(t=9.13-44.15, P<0.05). Transfection of si-FOXD1 effectively inhibited the expression of FOXD1 (t=10.51, P<0.05), decreased proliferation (t=10.41, P<0.05), increased radiosensitivity with a radiosensitization ratio of 1.797, and reduced the radiation-induced DNA-PK activity (t=6.20, P<0.05) in colorectal cancer cells. After localized irradiation, the tumor volume and weight in nude mice transplanted with si-FOXD1 HCT116 cells were significantly smaller than those in HCT116 (t=11.29, 3.69, P<0.05). Conclusions Knock-down of FOXD1 gene increases the radiosensitivity of colorectal cancer cells and inhibits the growth of colorectal cancer xenograft in nude mice, which provides a potential target gene in improving the effect of radiotherapy on colorectal cancer. Key words: FOXD1 gene; Colorectal cancer; Radiosensitiviy

The aim of this study was to explore the effect of long non-coding RNA TRIM52 antisense RNA 1 on the proliferation, apoptosis and radiosensitivity of colorectal cancer cells and its possible mechanism. Real-time quantitative reverse transcription polymerase chain reaction method was used to detect the expression of long non-coding RNA TRIM52 antisense RNA 1 and microRNA-361-5p in adjacent tissues, colorectal cancer tissues and human normal colorectal mucosal cell lines, fetal human cells and human colorectal cancer cell lines. The transplanted tumor experiment in nude mice was used to detect the influence of the expression changes of long non-coding RNA TRIM52 antisense RNA 1 and microRNA-361-5p on the transplanted tumor growth. The expression of long non-coding RNA TRIM52 antisense RNA 1 in colorectal cancer tissues and cell lines was increased, while the expression of microRNA-361-5p was decreased (p<0.05). After transfection of small interfering long non-coding RNA TRIM52 antisense RNA 1 or microRNA-361-5p mimics, cell viability and cell survival score were decreased, the number of cell clone formation was decreased and cell apoptosis rate was increased (p<0.05). Transfection of small interfering long non-coding RNA TRIM52 antisense RNA 1 could inhibit the growth of transplanted tumors in nude mice. Long non-coding RNA TRIM52 antisense RNA 1 could target the microRNA-361-5p expression. The expression of long non-coding RNA TRIM52 antisense RNA 1 could inhibit colorectal cancer cell proliferation and clone formation and promote cell apoptosis by targeted regulation of microRNA-361-5p expression and could enhance the radiosensitivity of colorectal cancer cells.

Objective:In this research we evaluated molecular mechanism of effect of metformin in radio sensitivity of breast cancer cells. Methods:This research was done in cellular and molecular research center of Qazvin university of Medical science in 1399 to 1401. Studied samples were two breast cancer cell lines (MCF-7 and MDA-MB-231) they are derived from primary and secondary tumors resected from a single patient. We exposed them to cumulative 50 Gy radiation and constructed radio resistant cell lines. Then resistant cell lines were treated with 50µm of metformin. Our studied groups were resistant cells treated and un treated with metformin. Then, the expression rate of miR-21-5p and SESN1 gene in resistant and control cells was checked by Quantitative Real-time PCR(qRTPCR). After the cell lines were treated with different concentrations of metformin at different intervals, the rate of cell proliferation and cell death was checked by CCK-8 assay and flow cytometry. The Western blot method was also used to confirm the expression of some genes.Results:Our results showed that the expression of miR-21-5p was upregulated in radiation-resistant cancer cells (1.8±0.65) (P<0.0001) MCF-7 cell line and (1.6±0.42)(P<0.001) MBA-MD-231 cell line, while the expression of SESN1 was down regulated (0.46±0.12) (P<0.0001) MCF-7 cell line and (0.42±0.22) (P<0.001) MBA-MD-231 cell line. Metformin enhanced the radio sensitivity of breast cancer cells in a dose and time-dependent manner. Also, metformin treatment decreased the expression of miR-21-5p (0.47±0.32) (P<0.0001) MCF-7 Cell line and (0.45±0.21)(P<0.001) MBA-MD-231 cell line and increased the expression of SESN1 (1.65±0.72)(P<0.0001)MCF-7 cell line and (1.73±0.52)(P<0.0001) MBA-MD-231 cell line. The function of metformin was reversed by miR-21-5p inhibitors or the transfection of SESN1 overexpressing plasmids. Conclusion:In conclusion, based on this research results, metformin enhanced the radio sensitivity of breast cancer cells via modulating the expression of miR-21-5p and SESN1.

Tumor‑associated macrophages (TAMs), a major component of the tumor microenvironment, are crucial to the processes of tumor growth, infiltration and metastasis, and contribute to drug resistance. The importance of TAMs in radiation resistance of colorectal cancer remains unclear. To investigate the effects of autophagy regulation of TAMs on the radiosensitivity of colorectal cancer cells, the current study induced TAM formation from THP‑1 monocyte cells. Sequential treatment of THP‑1 cells with PMA for 72 h and human recombinant interleukin‑4 for 24 h was used to stimulate THP‑1 differentiation to TAMs. Expression of the cell surface markers CD68, CD204 and CD206, and changes to cell morphology were used to confirm successful differentiation. The TAMs were stimulated to promote or inhibit autophagy during co‑culture with LoVo colorectal adenocarcinoma cells. The cells were irradiated, with subsequent measurement of LoVo colony formation and apoptosis. Additionally, the expression of p53, Bcl‑2, survivin and Smac proteins was assessed by western blotting. Monodansylcadaverin staining was used to analyze the presence of autophagic vacuoles in TAM, and western blot analysis was used to assess the expression of Beclin‑1, LC3B I and II, ATG‑3, ‑5 and ‑7. The results demonstrated TAM autophagy to be markedly altered by rapamycin and bafilomycin A1 treatment. Following co‑culture with TAMs, the colony formation rate and survival fraction of LoVo cells were significantly higher than those in the control group (P<0.05). It was further demonstrated that the regulation of autophagy in TAMs was able to inhibit the colony formation of LoVo colorectal cancer cells. Upregulation of TAM autophagy using rapamycin exhibited more effective inhibition of LoVo colony formation than autophagy downregulation. Notably, apoptosis was significantly increased in LoVo cells when co‑cultured with TAMs only, or with rapamycin‑mediated autophagy upregulated TAMs, compared with LoVo cells cultured alone (P<0.01). Expression of Bcl‑2, survivin and p53 were reduced in LoVo cells co‑cultured with TAMs, compared with the control group (P<0.05), whereas Smac expression was increased in the co‑culture groups (P<0.01). It was demonstrated that rapamycin‑mediated autophagy stimulation in TAMs led to reduced expression levels of survivin and Bcl‑2, however, Smac expression was increased. The upregulation of autophagy in TAMs inhibited proliferation and induced apoptosis in colon cancer cells, and altered the expression of radiosensitivity‑associated proteins. This data indicated that the radiosensitivity of colorectal cancer cells is associated with autophagy of TAM, and that stimulating TAM autophagy may increase the radiosensitivity of colorectal cancer cells.

Objective: To investigate the effect of circBANP on radiosensitivity of colorectal cancer cells and subcutaneous transplanted tumor in nude mice and its potential molecular mechanism. Methods: The carcinoma and adjacent normal mucosal tissues of 20 patients with colorectal cancer who were surgically resected in Henan People's Hospital from January 2018 to January 2019 were selected. The radio-resistant colorectal cancer cell LoVo/R was established. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expressions of circBANP and miR-338-3p. The radiation sensitivity was determined by cell clone formation experiment. Cell vitality was detected by using methyl thiazolyl tetrazolium (MTT). The expressions of autophagy-related protein microtubule-associated protein light chain 3 (LC3) and p62 were detected by western blot. The fluorescence intensity of LC3 in cells was detected by immunofluorescence assay. The downstream microRNAs (miRNAs) of circBANP were predicted by Circular RNA Interactome website and further verified by dual luciferase reporter gene assay. The transplanted tumor model of LoVo/R cells in nude mice was established, and the effect of circBANP on the growth of transplanted tumor after radiation was observed. Results: The expression levels of circBANP and miR-338-3p in colorectal cancer tissues were 3.21+ 0.29 and 0.47+ 0.04, respectively, which were significantly higher than 1.00+ 0.07 and 1.00+ 0.05 in adjacent tissues (P<0.05). The circBANP expression level of LoVo/R cells was 3.21±0.34, higher than 1.00±0.07 of LoVo cells (P<0.05), and the expression level of miR-338-3p of LoVo/R cells was 0.33±0.04, lower than 1.00±0.08 of LoVo cells (P<0.05). After 4 Gy irradiation, compared with the control group, the viability of LoVo/R cells in the circBANP silencing group [(34±4)% vs (62±6)%, P<0.05], the cell survival fraction (0.07±0.02 vs 0.27±0.04, P<0.05) were decreased, and the radiation sensitization ratio was 1.843, the expression of LC3Ⅱ/Ⅰin LoVo/R cells increased while p62 expression decreased, the cell autophagy was observed. Autophagy inhibitor chloroquine reversed the increased expression of LC3Ⅱ/Ⅰ and inhibited expression of p62 in LoVo/R cells induced by radiation, and promoted the suppression of cell viability and survival induced by radiation, the radiotherapy sensitization ratio was 1.780. Compared with control group after 4 Gy irradiation, the relative fluorescence intensity of LC3 in circBANP silencing LoVo/R cells decreased (0.11±0.01 vs 1.00±0.12, P<0.05), the expression of LC3-Ⅱ/Ⅰdecreased (1.25±0.13 vs 3.84±0.39, P<0.05) while p62 expression increased (2.76±0.29 vs 1.00±0.08, P<0.05). As predicted by Circular RNA Interactome website and confirmed by double luciferase reporter gene assay, miR-338-3p was the target gene of circBANP. The relative fluorescence intensity of LC3 in circBANP silencing + anti-miR-338-3p + 4 Gy group increased (7.32±0.72 vs 1.00±0.09, P<0.05), the expression level of LC3-Ⅱ/Ⅰ increased (4.13±0.43 vs 2.31±0.23, P<0.05) while p62 expression decreased (0.34±0.03 and 1.00±0.11, P<0.05), the radiotherapy sensitization ratio was 0.596. Nude mice subcutaneously transplanted tumor experiment showed that the tumor volume and weight of circBANP silencing group on 13, 16, 19, 22, 25, 28, and 31 days were lower than those of control group (P<0.05), while the tumor volume and weight of circBANP silencing + anti-miR-338-3p group on days of 13, 16, 19, 22, 25, 28 and 31 after inoculated were higher than those of circBANP+ anti-miR-NC group (P<0.05). Conclusions: CircBANP can regulate the radiosensitivity of colorectal cancer cells by regulating the expression of miR-338-3p, and affect the growth of transplanted tumor in nude mice. CircBANP may be a potential target for enhancing radiosensitivity of colorectal cancer cells.

The PI3K/AKT/mTOR pathway and autophagy are known to play important roles in cancer radioresistance. The aim of the present study was to investigate whether the combination of temsirolimus (TEM), an mTOR inhibitor, and chloroquine(CQ), an autophagy inhibitor, can increase radiosensitivity in colorectal cancer(CRC) cells. The efficacies of TEM and/or CQ as radiosensitizers were examined using clonogenic assays in CRC cell lines SW480 and HT‑29. The expression levels of the phosphorylated isoforms of S6 and 4E‑BP1, downstream proteins of mTOR, as well as the expression levels of p62 and LC3, autophagy‑related proteins, were assessed by western blot analysis. The formation of acidic organelles was detected in acridine orange‑stained cells. Apoptosis and caspase activity were assessed using flow cytometry. The results revealed that ionizing radiation(IR) activated the downstream proteins of mTOR and induced autophagy. In the clonogenic assays, neither TEM nor CQ influenced the efficacy of IR, whereas their combination significantly increased the dose‑dependent efficacy of IR. TEM inhibited phosphorylation of the downstream proteins of mTOR and induced autophagy. CQ inhibited autophagy in the late phase and did not influence the downstream proteins of mTOR. TEM and CQ inhibited both the phosphorylation of downstream proteins of mTOR and autophagy. Cell death analysis revealed that the combination of TEM and CQ strongly induced apoptosis in cells exposed to IR. In conclusion, the combination of TEM and CQ increased radiosensitivity in CRC cells through co‑inhibition of mTOR and autophagy.

ObjectiveTo propose and verify that miRNA‐31 increases the radiosensitivity of colorectal cancer and explore its potential mechanism.MethodA bioinformatics analysis was performed to confirm that the expression of miRNA‐31 was higher in colorectal cancer than in normal colorectal tissue. The expression of miRNA‐31 was detected to verify the change in its expression in a radiotherapy‐resistant cell line. Methylation was detected to explore the cause of the decrease in miRNA‐31 expression. Overexpression or inhibition of miRNA‐31 further confirmed the change in its expression in colorectal cancer cell lines. Bioinformatics methods were used to screen the downstream target genes and for experimental verification. A luciferase assay was performed to determine the miRNA‐31 binding site in STK40. Overexpression or inhibition of STK40 in colorectal cancer cell lines further confirmed the change in STK40 expression in vitro.ResultsThe bioinformatics results showed higher expression of miRNA‐31 in tumors than in normal tissue, and miRNA‐31 mainly participated in the pathway related to cell replication. Next, we observed the same phenomenon: miRNA‐31 was expressed at higher levels in colorectal tumors than in normal colorectal tissue and its expression decreased in radiation‐resistant cell lines after radiation, implying that miRNA‐31 increased the radiosensitivity of colorectal cancer cell lines. No significant change in upstream methylation was observed. miRNA‐31 regulated the radiosensitivity of colorectal cancer cell lines by inhibiting STK40. Notably, miRNA‐31 played a role by binding to the 3′ untranslated region of SK40. STK40 negatively regulated the radiosensitivity of colorectal cancer cells.ConclusionsmiRNA‐31 increases the radiosensitivity of colorectal cancer cells by targeting STK40; miRNA‐31 and STK40 are expected to become potential biomarkers for increasing the sensitivity of tumor radiotherapy in clinical treatment.

Objective To study the effect of LncRNA CRNDE on radiosensitivity of colorectal cells and underlying mechanism. Methods Colorectal cancer HT-29 cells were transfected with CRNDE shRNA and the interference efficiency was determined by Real time PCR. HT-29 cells transfected with CRNDE shRNA or co-transfected with CRNDE shRNA and miR-384 inhibitor were irradiated at 8 Gy dose, then cell proliferation and apoptosis were detected by MTT and flow cytometry assay, respectively, and cell radiosensitivity was evaluated by cloning assay. It was predicted by a bioinformatics software that CRNDE and miR-384 have complementary binding sites, and this was identified by a luciferase reporting system. Results CRNDE shRNA reduced the expression of CRNDE in HT-29 cells(1.00±0.08 vs. 0.42±0.06, t=10.051, P<0.05). Both CRNDE shRNA and radiation inhibited the proliferation and induced apoptosis of HT-29 cells, and their combination treatment had synergistic effect in apoptosis induction [Apoptosis rates: (2.27±0.13)%, (23.58±2.35)%, (26.91±2.81)%, (36.84±3.24)%, F=24.660, P<0.05; A values: 0.45±0.06, 0.30±0.02, 0.28±0.03, 0.20±0.02, F=106.207, P<0.05]. Transfection of CRNDE shRNA increased the radiosensitivity of HT-29 cells with a radiosensitization ratio of 1.374. CRNDE negatively regulated the expression of its target miR-384. The miR-384 inhibitor antagonized the effect of CRNDE shRNA on proliferation inhibition and apoptosis promotion of radiation-treated colorectal cancer cells. Conclusions Down-regulation of LncRNA expression enhances the radiosensitivity of colorectal cancer cells by regulating miR-384 expression. Key words: Colorectal cancer; Colorectal neoplasia differentially expressed(CRNDE); miR-384; Radiosensitivity

Objective To investigate the correlation between the level of apoptosis protein 1 (c-IAP1) and the radiosensitivity of lung cancer cells. Method The survival rate and proliferation of the lung cancer cells lines (A549, H460, H1299, H358, HCC827, H1650) from six human were detected by thiazolyl blue tetrazolium bromide (MTT) and cell colony formation assay. The DNA damage effects of radiation on lung cancer cells were detected by comet assay. The expressions of c-IAP1 protein and its mRNA were determined by Western blot and real-time quantitative PCR. Results The results of MTT and colony formation showed that the radiosensitivity of different lung cancer cells was also different, among which H358 and H460 cells had the highest radiosensitivity than that of H1650 and HCC827 cells, and H1299 and A549 cells had the weakest radiosensitivity. The results of comet assay showed that six kinds of lung cancer cells were suffered by DNA damage after radiation, and the DNA damage of H358 cells was most serious. The results of Western blot and real-time quantitative PCR showed that the c-IAP1 protein level was negatively correlated with the radiosensitivity of lung cancer cells. The higher the c-IAP1 protein level, the weaker the radiosensitivity of cells. The radiosensitivity was also affected by Smac protein levels. Conclusions c-IAP1 may be a selective target gene in mediating the radiosensitivity of lung cancer cells and this paper may contribute to the study of radioresistance and radiosensitization of cancer cell. Key words: Cellular inhibitor of apoptosis protein 1; Radiosensitivity; Lung cancer cells; γ-ray

BACKGROUND Colorectal cancer (CRC), the third most common cancer worldwide, has increasingly detrimental effects on human health. Radiotherapy resistance diminishes treatment efficacy. Studies suggest that spermine synthase (SMS) may serve as a potential target to enhance the radiosensitivity. AIM To investigate the association between SMS and radiosensitivity in CRC cells, along with a detailed elucidation of the underlying mechanisms. METHODS Western blot was adopted to assess SMS expression in normal colonic epithelial cells and CRC cell lines. HCT116 cells were transfected with control/SMS-specific shRNA or control/pcDNA3.1-SMS plasmids. Assessments included cell viability, colony formation, and apoptosis via MTT assays, colony formation assays, and flow cytometry. Radiosensitivity was studied in SMS-specific shRNA-transfected HCT116 cells post-4 Gy radiation, evaluating cell viability, colony formation, apoptosis, DNA damage (comet assays), autophagy (immunofluorescence), and mammalian target of rapamycin (mTOR) pathway protein expression (western blot). RESULTS Significant up-regulation of SMS expression levels was observed in the CRC cell lines. Upon down-regulation of SMS expression, cellular viability and colony-forming ability were markedly suppressed, concomitant with a notable increase in apoptotic indices. Furthermore, attenuation of SMS expression significantly augmented the sensitivity of HCT116 cells to radiation therapy, evidenced by a pronounced elevation in levels of cellular DNA damage and autophagy. Importantly, down-regulation of SMS corresponded with a marked reduction in the expression levels of proteins associated with the mTOR signaling pathway. CONCLUSION Knocking down SMS attenuates the mTOR signaling pathway, thereby promoting cellular autophagy and DNA damage to enhance the radiosensitivity of CRC cells.

To screen long non-coding RNA (lncRNA) associated with radiosensitivity in colorectal carcinoma cell lines. Colony formation assay was performed in colorectal cancer cell lines HT29, SW480, RKO, Lovo and HCT116 after irradiation with different radiation doses. Radiation sensitivity of these 5 cell lines was detected through survival fraction at 2 Gy (SF2 value). High-throughput lncRNA chip was used to screen lncRNA genes with expression differences more than 2 folds among SW480, RKO and Lovo. Further experiment on the expression differences of lncRNAs selected was conducted by realtime PCR. The radiosensitivity order of these 5 cell lines from low to high (SF2 value from high to low) was HT29 (0.83 ± 0.03), SW480 (0.69 ± 0.02), RKO(0.53 ± 0.02), Lovo (0.47 ± 0.05), HCT116 (0.32 ± 0.03) (P < 0.01). Five lncRNAs associated with radiation sensitivity were screened. Among them, expression levels of R05532, NR_015441, and NR_033374 were positively correlated with radiation resistance(all P < 0.01), and expression levels of the other 2 lncRNAs, NR_073156 and AA745020, were not correlated with radiation resistance of colorectal cancer cells (both P>0.05). lncRNA R05532, NR_015441 and NR_033374 may be used as the predictive marker of radiosensitivity of colorectal cancer cells. Higher expression of these genes shows radiation resistance.

Objective To investigate the effect of DDX46 gene on the radiosensitivity of colorectal cancer cells and underlying mechanism. Methods SW480 cells transfected with DDX46 RNAi or its empty plasmid by lentivirus were set as experimental group and control group, respectively. After 72 h of transfection, the cells were irradiated with 4 Gy X rays. The cell viabilities of these two groups were detected by CCK-8 assay. The number of γ-H2AX foci and the expressions of some key proteins related to DNA damage repair were detected by immunofluorescence technique and Western blot at 24 h after irradiation. Results At 24 h after 4 Gy irradiation, the cell vitality of experimental group was decreased to(15.02±3.92)%(t=-4.696, P<0.05)of control and(17.43±1.83)%(t=4.844, P<0.01) of nonirradiated cells, but there was no significant difference between 4 Gy irradiated control cells and nonirradiated cells. Meanwhile, compared with the control group, the ATM protein expression level (t=7.530, P<0.01) and the number of γ-H2AX foci (t=-3.108, P<0.05) were significantly increased in the experimental SW480 cells, while the expression levels of p-ATM and Rad50 were significantly reduced (t=4.260, 4.260, P<0.05), and the protein levels of DNA-PK in these two groups had tiny difference. Conclusions DDX46 RNA silence increases the radiation sensitivity of SW480 cells by inhibiting ATM activation and DNA repair. Key words: Colorectal cancer cells(CRC); DDX46; Radiosensitivity

Objective To investigate the effect and mechanism of LncRNA ANRIL on the radiosensitivity of HCT116 cells line and nude mouse transplant tumors. Methods The expression of LncRNA ANRIL in colorectal cancer cells was detected by qPCR. The negative control siRNA, ANRIL siRNA, miR-NC mimic, miR-195 mimic, miR-NC inhibitor and miR-195 inhibitor were transfected into HCT116 cells, and marked as negative control group, silencing ANRIL group, overexpressing miR-NC group, overexpressing miR-195 group, inhibiting miR-NC group and inhibiting miR-195 group, and the HCT116 cells without any treatment were marked as the blank control group. The clone formation assay was used to detect radiosensitivity of colorectal cancer cells, flow cytometry was used to detect apoptosis. The web site, StarBase, was used to predict the downstream miRNAs of ANRIL and dual luciferase reporter gene assay was used to further verify. Subcutaneous tumor transplantation assay was used to detect the effect of ANRIL on the growth of colorectal cancer cells after irradiation. Results After irradiation with 2, 4, 6 and 8 Gy, the cell survival fraction of silencing ANRIL group was significantly decreased when compared with that of negative control group (P<0.05), and the radiosensitivity ratio was 1.52. The apoptosis rate of the silencing ANRIL+ 4 Gy group was significantly higher than that of the negative control+ 4 Gy group ((27.86±2.78)% vs. (12.06±1.46)%, P<0.05). The results of the experiment on nude mouse transplant tumors showed that the tumor volume in the negative control group was lower than that of the silent ANRIL group on days 13, 16, 19, 22 and 25 ((234±66) mm3, (273±63) mm3, (296±72) mm3, (321±85) mm3 and (403±94) mm3vs. (357±79) mm3, (485±124) mm3, (617±143) mm3, (764±174) mm3 and (985±221) mm3P<0.05). MiR-195 is a target gene of ANRIL, and inhibition of miR-195 can reverse the inhibitory effect of silencing ANRIL on radiosensitivity, apoptosis and xenografts of HCT116 cells. Conclusions LncRNA ANRIL regulates the radiosensitivity of colorectal cancer cells by miR-195, which may provide a new sensitizing target for clinical colorectal cancer radiotherapy. Key words: ANRIL gene; miR-195 gene; HCT116 cell line; Nude mouse transplant tumors; Radiosensitivity

BackgroundmicroRNAs (miRNAs) can regulate the sensitivity of cancer cells to chemotherapy and radiotherapy. Aberrant expression of miR-195 has been found to be involved in colorectal cancer (CRC); however, its function and underlying mechanism in the radioresistance of CRC remains unclear.MethodsThe levels of miR-195 and CARM1 were detected by quantitative reverse transcription-polymerase chain reaction and Western blot analysis in HCT-116 and HT-29 cells, respectively. Colony survival and apoptosis were determined by clonogenic assay and flow cytometry analysis, respectively. The apoptosis-related proteins Bax, Bcl-2, and γ-H2AX were detected using Western blot. The targets of miR-195 were identified by bioinformatic prediction and luciferase reporter assays. CRC cells in vitro and in vivo were exposed to different doses of X-ray radiations.ResultsmiR-195 was downregulated, and CARM1 was upregulated in HCT-116 and HT-29 cells. miR-195 overexpression or CARM1 knockdown suppressed colony survival, induced apoptosis, promoted expression of Bax and γ-H2AX, and inhibited Bcl-2 expression in CRC cells. CARM1 was identified and validated to be a functional target of miR-195. Moreover, restored expression of CARM1 reversed the enhanced radiosensitivity of CRC cells induced by miR-195. Furthermore, miR-195 increased the sensitivity of CRC cells to radiation in vivo.ConclusionmiR-195 enhances radiosensitivity of CRC cells through suppressing CARM1. Therefore, miR-195 acts as a potential regulator of radioresistance for CRC cells and as a promising therapeutic target for CRC patients.

Objective Toll-like receptor 4 (TLR4) is involved in tumor development. Numerous studies have confirmed that TLR4 mediates processes in tumorigenesis, for example, inflammation, proliferation and invasion. However, the effects of TLR4 on colorectal cancer development have not been fully elucidated. The present study aimed to evaluate the effects and mechanisms of TLR4 on colorectal cancer development. Methods The expression of TLR4 and acyl coenzyme A cholesterol acyltransferase 1 (ACAT1) in colorectal cancer tissues and cell lines was detected using reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. Cell counting kit-8 (CCK-8) and Transwell assays were used to evaluate the effects of TLR4 silencing on cell proliferation, migration and invasion in HT29 and SW480. RT-PCR and Western blotting were used to determine the regulation between TLR4 and ACAT1. Results Both TLR4 and ACAT1 were highly expressed in colorectal cancer tissues (TLR4 mRNA: 2.395 0±0.247 7 and 1.093 0±0.075 8, P=0.000; ACAT1 mRNA: 2.196 0±0.189 5 and 1.093 0±0.075 8, P=0.000) and cell lines. Inhibition of TLR4 suppressed cell proliferation, migration and invasion in HT29 and SW480. TLR4 small interfering RNA(siRNA) decreased ACAT1 expression in HT29 (mRNA: 0.350 0±0.145 7 and 1.017 0±0.145 0, P=0.032; Protein: 0.523 3±0.052 1 and 1.140 0±0.105 8, P=0.006), and that overexpression of ACAT1 by pLV-Neo-ACAT1 abolished the effects of TLR4 siRNA on colorectal cancer cell lines. Conclusion TLR4 siRNA inhibits cell proliferation, migration and invasion by suppressing ACAT1 expression, suggesting that TLR4 may be a potential therapeutic target for the treatment of colorectal cancer. Key words: Toll-like receptor 4; Small interfering RNA; Colorectal cancer; Acyl coenzyme A cholesterol acyltransferase 1